Methods of Improving the Introduction of DNA Into Bacterial Cells

ABSTRACT

The present invention relates to methods of improving the introduction of DNA into bacterial host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/896,098, filed Oct. 1, 2010, which is a divisional of U.S. patentapplication Ser. No. 12/516,438, now U.S. Pat. No. 7,820,408, which is a35 U.S.C. 371 National Stage Application of PCT/US2007/085840, filed onNov. 29, 2007, which claims priority from U.S. Provisional PatentApplication No. 60/861,896, filed on Nov. 29, 2006. The content of theseapplications are fully incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biologicalmaterial, which deposits are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of improving the introductionof DNA into bacterial host cells.

2. Description of the Related Art

Type II restriction endonucleases are reportedly effective barriers tothe introduction of DNA into bacteria (Briggs et al., 1994, Applied andEnvironmental Microbiology 60: 2006-2010; Accetto et al., 2005, FEMSMicrobiology Letters 247: 177-183). Numerous Type II restrictionendonucleases have been characterized in Bacillus and many commerciallyavailable restriction endonucleases have been isolated from Bacillusspecies (Roberts, et al., 2005, Nucleic Acids Research 33: 230-232).

Host DNA is protected from cleavage by its native restrictionendonuclease due to host DNA modification by a corresponding DNAmethyltransferase. The restriction endonuclease and DNAmethyltransferase genes usually lie adjacent to each other in the genomeand constitute a restriction-modification (R-M) system. These genes maybe oriented transcriptionally in a convergent, divergent, or sequentialmanner. Although restriction endonucleases have little if any sequencesimilarity between one another, a limited amino acid motif, PD . . .D/EXK, has been found in many restriction endonucleases (Pingoud andJeltsch, 2001, Nucleic Acids Research 29: 3705-3727). In contrast,several general motifs have been found for the DNA methyltransferases(Kumar et al., 1994, Nucleic Acids Research 22: 1-10; Smith et al.,1990, Proceedings of the National Academy of Sciences USA 87: 826-830),which has allowed identification of restriction endonucleases by firstidentifying their more homologous corresponding DNA methyltransferases.

The introduction of DNA into a bacterial host cell, e.g., Bacilluslicheniformis, can be an inefficient process, resulting in few, if any,transformants. There is a need in the art for new methods of introducinga DNA into a bacterial host cell to improve the efficiency of obtainingtransformants with the DNA.

The present invention relates to improved methods of introducing DNAinto a bacterial host cell.

SUMMARY OF THE INVENTION

The present invention relates to isolated polynucleotides encoding DNAmethyltransferases selected from the group consisting of (a) apolynucleotide encoding a polypeptide comprising an amino acid sequencehaving at least 60% sequence identity with amino acids 1 to 337 of SEQID NO: 2; (b) a polynucleotide comprising a nucleotide sequence havingat least 60% sequence identity with nucleotides 1 to 1011 of SEQ ID NO:1; (c) a polynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-lengthcomplementary strand; and (d) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 337 of SEQ ID NO: 2.

The present invention also relates to isolated DNA methyltransferasesselected from the group consisting of (a) a polypeptide comprising anamino acid sequence having at least 60% sequence identity with aminoacids 1 to 337 of SEQ ID NO: 2; (b) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1; (c) apolypeptide encoded by a polynucleotide that hybridizes under at leastmedium stringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1or its full-length complementary strand; and (d) a variant comprising asubstitution, deletion, and/or insertion of one or more amino acids ofamino acids 1 to 337 of SEQ ID NO: 2.

The present invention also relates to isolated polynucleotides encodingrestriction endonucleases selected from the group consisting of (a) apolynucleotide encoding a polypeptide comprising an amino acid sequencehaving at least 60% sequence identity with amino acids 1 to 381 of SEQID NO: 4; (b) a polynucleotide comprising a nucleotide sequence havingat least 60% sequence identity with nucleotides 1 to 1143 of SEQ ID NO:3; (c) a polynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1143 of SEQ ID NO: 3 or its full-lengthcomplementary strand; and (d) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 381 of SEQ ID NO: 4.

The present invention also relates to isolated restriction endonucleasesselected from the group consisting of (a) a polypeptide comprising anamino acid sequence having at least 60% sequence identity with aminoacids 1 to 381 of SEQ ID NO: 4; (b) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1143 of SEQ ID NO: 3; (c) apolypeptide encoded by a polynucleotide that hybridizes under at leastmedium stringency conditions with nucleotides 1 to 1143 of SEQ ID NO: 3or its full-length complementary strand; and (d) a variant comprising asubstitution, deletion, and/or insertion of one or more amino acids ofamino acids 1 to 381 of SEQ ID NO: 4.

The present invention also relates to methods of producing bacterialtransformants, comprising:

(a) introducing a DNA into a first bacterial host cell comprising apolynucleotide encoding a DNA methyltransferase to methylate the DNA;

wherein the polynucleotide encoding the DNA methyltransferase isselected from the group consisting of (i) a polynucleotide encoding apolypeptide comprising an amino acid sequence having at least 60%sequence identity with amino acids 1 to 337 of SEQ ID NO: 2; (ii) apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1; (iii) apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-lengthcomplementary strand; and (iv) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 337 of SEQ ID NO: 2; and

wherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2;

(b) transferring the methylated DNA from the first bacterial host cellinto a second bacterial host cell; wherein the second bacterial hostcell comprises a restriction endonuclease able to degrade the DNA butunable to degrade the methylated DNA; and

(c) isolating transformants of the second bacterial host cell comprisingthe methylated DNA.

The present invention also relates to methods of producing bacterialtransformants, comprising:

(a) methylating in vitro a DNA with a DNA methyltransferase to produce amethylated DNA;

wherein the DNA methyltransferase is selected from the group consistingof (i) a polypeptide comprising an amino acid sequence having at least60% sequence identity with amino acids 1 to 337 of SEQ ID NO: 2; (ii) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 60% sequence identity with nucleotides 1 to 1011 of SEQID NO: 1; (iii) a polypeptide encoded by a polynucleotide thathybridizes under at least medium stringency conditions with nucleotides1 to 1011 of SEQ ID NO: 1 or its full-length complementary strand; and(iv) a variant comprising a substitution, deletion, and/or insertion ofone or more amino acids of amino acids 1 to 337 of SEQ ID NO: 2; and

wherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2;

(b) introducing the methylated DNA into a bacterial host cell, whereinthe bacterial host cell comprises a restriction endonuclease able todegrade the DNA but unable to degrade the methylated DNA; and

(c) isolating transformants of the bacterial host cell comprising themethylated DNA.

The present invention also relates to methods of producing bacterialtransformants, comprising:

(a) introducing a DNA into a bacterial host cell comprising apolynucleotide encoding a restriction endonuclease, which isinactivated;

wherein the polynucleotide encoding the restriction endonuclease isselected from the group consisting of (i) a polynucleotide encoding apolypeptide comprising an amino acid sequence having at least 60%sequence identity with amino acids 1 to 381 of SEQ ID NO: 4; (ii) apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1143 of SEQ ID NO: 3; (iii) apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1143 of SEQ ID NO: 3 or its full-lengthcomplementary strand; and (iv) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 381 of SEQ ID NO: 4;

wherein the restriction endonuclease has the same specificity as therestriction endonuclease of amino acids 1 to 381 of SEQ ID NO: 4; and

wherein the inactivation of the polynucleotide encoding the restrictionendonuclease prevents the introduced DNA from being digested by therestriction endonuclease and avoids the need to methylate the DNA with aDNA methyltransferase prior to introducing the DNA into the bacterialhost cell; and

(b) isolating transformants of the bacterial host cell comprising theDNA.

The present invention also relates to methods of producing a polypeptidehaving biological activity, comprising:

(a) cultivating a bacterial host cell comprising an introduced DNAencoding or involved in the expression of the polypeptide havingbiological activity under conditions conducive for production of thepolypeptide;

wherein the DNA is methylated prior to being introduced into thebacterial host cell by a DNA methyltransferase selected from the groupconsisting of (i) a polypeptide comprising an amino acid sequence havingat least 60% sequence identity with amino acids 1 to 337 of SEQ ID NO:2; (ii) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 60% sequence identity withnucleotides 1 to 1011 of SEQ ID NO: 1; (iii) a polypeptide encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-lengthcomplementary strand; and (iv) a variant comprising a substitution,deletion, and/or insertion of one or more amino acids of amino acids 1to 337 of SEQ ID NO: 2;

wherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2; and

wherein the methylation prevents the introduced DNA from being digestedby a restriction endonuclease of the bacterial host cell; and

(b) recovering the polypeptide having biological activity.

The present invention also relates to methods of producing a polypeptidehaving biological activity, comprising:

(a) cultivating a bacterial host cell comprising an introduced DNAencoding or involved in the expression of the polypeptide havingbiological activity under conditions conducive for production of thepolypeptide;

wherein the bacterial host cell comprises a polynucleotide encoding arestriction endonuclease, which is inactivated;

wherein the polynucleotide encoding the restriction endonuclease isselected from the group consisting of (i) a polynucleotide encoding apolypeptide comprising an amino acid sequence having at least 60%sequence identity with amino acids 1 to 381 of SEQ ID NO: 4; (ii) apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1143 of SEQ ID NO: 3; (iii) apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1143 of SEQ ID NO: 3 or its full-lengthcomplementary strand; and (iv) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 381 of SEQ ID NO: 4;

wherein the restriction endonuclease has the same specificity as therestriction endonuclease of amino acids 1 to 381 of SEQ ID NO: 4; and

wherein the inactivation of the polynucleotide encoding the restrictionendonuclease prevents the introduced DNA from being digested by therestriction endonuclease and avoids the need to methylate the DNA with aDNA methyltransferase prior to introducing the DNA into the bacterialhost cell; and

(b) recovering the polypeptide having biological activity.

The present invention also relates to the bacterial host cells describedabove.

The present invention also relates to methods of producing a mutant of aparent bacterial cell, comprising:

(a) introducing into a parent bacterial cell a DNA comprising a nucleicacid construct to inactivate a gene encoding a polypeptide in the parentbacterial cell, which results in a mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions;

wherein the bacterial host cell comprises a polynucleotide encoding arestriction endonuclease, which is inactivated;

wherein the polynucleotide encoding the restriction endonuclease isselected from the group consisting of (i) a polynucleotide encoding apolypeptide comprising an amino acid sequence having at least 60%sequence identity with amino acids 1 to 381 of SEQ ID NO: 4; (ii) apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity with nucleotides 1 to 1143 of SEQ ID NO: 3; (iii) apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1143 of SEQ ID NO: 3 or its full-lengthcomplementary strand; and (iv) a polynucleotide encoding a variantcomprising a substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 381 of SEQ ID NO: 4;

wherein the restriction endonuclease has the same specificity as therestriction endonuclease of amino acids 1 to 381 of SEQ ID NO: 4; and

wherein the inactivation of the polynucleotide encoding the restrictionendonuclease prevents the introduced DNA from being digested by therestriction endonuclease and avoids the need to methylate the DNA with aDNA methyltransferase prior to introducing the DNA into the parentbacterial cell; and

(b) isolating the mutant cell.

The present invention also relates to methods of producing a mutant of aparent bacterial cell, comprising:

(a) introducing into a parent bacterial cell a DNA comprising a nucleicacid construct to inactivate a gene encoding a polypeptide in the parentbacterial cell, which results in a mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions;

wherein the DNA is methylated prior to being introduced into thebacterial host cell by a DNA methyltransferase selected from the groupconsisting of (i) a polypeptide comprising an amino acid sequence havingat least 60% sequence identity with amino acids 1 to 337 of SEQ ID NO:2; (ii) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 60% sequence identity withnucleotides 1 to 1011 of SEQ ID NO: 1; (iii) a polypeptide encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or its full-lengthcomplementary strand; and (iv) a variant comprising a substitution,deletion, and/or insertion of one or more amino acids of amino acids 1to 337 of SEQ ID NO: 2;

wherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2; and

wherein the methylation prevents the introduced DNA from being digestedby a restriction endonuclease of the parent bacterial cell; and

(b) isolating the mutant cell.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of a Bacillus licheniformis M.Bli1904II DNA methyltransferase(SEQ ID NOs: 1 and 2, respectively).

FIGS. 2A and 2B show the genomic DNA sequence and the deduced amino acidsequence of a Bacillus licheniformis Bli1904II restriction endonuclease(SEQ ID NOs: 3 and 4, respectively).

FIG. 3 shows the genomic DNA sequence of a Bacillus licheniformisBli1904II restriction-modification system comprising genes encodingBli1904II restriction endonuclease and M.Bli1904II DNA methyltransferase(SEQ ID NO: 5). Reverse complement of the Bli1904II restrictionendonuclease coding region is indicated by double underscoring and ofthe M.Bli1904II DNA methyltransferase coding region is indicated bysingle underscoring.

FIG. 4 shows a restriction map of pMDT138.

FIG. 5 shows a restriction map of pKK223-3.

FIG. 6 shows a restriction map of pNBT51.

FIG. 7 shows a restriction map of pNBT52.

FIG. 8 shows a restriction map of pNBT53.

FIG. 9 shows a restriction map of pNBT54.

FIG. 10 shows a restriction map of pNBT35.

FIG. 11 shows a restriction map of pNBT30.

FIG. 12 shows a restriction map of pNBT31.

FIG. 13 shows a restriction map of pNBT36.

FIG. 14 shows a restriction map of pMDT100.

FIG. 15 shows a restriction map of pMDT156.

FIG. 16 shows a restriction map of pMDT134.

FIG. 17 shows a restriction map of pMDT131.

FIG. 18 shows a restriction map of pMDT139.

DEFINITIONS

M.Bli1904II DNA methyltransferase: The term “M.Bli1904II DNAmethyltransferase” is defined herein as a DNA(cytosine-5)-methyltransferase (EC 2.1.1.37) that catalyzes the transferof a methyl group from S-adenosyl-L-methionine to DNA within thesequence GCNGC, resulting in S-adenosyl-L-homocysteine and DNAcontaining 5-methylcytosine. For purposes of the present invention, DNAmethyltransferase activity is determined according to the proceduredescribed by Pfeifer et al., 1983, Biochim. Biophys. Acta 740: 323-30.One unit of DNA methyltransferase activity is the amount required toprotect 1 μg of λ DNA in 1 hour in a total reaction volume of 20 μlagainst cleavage by the corresponding restriction endonuclease.

The DNA methyltransferases of the present invention have at least 20%,preferably at least 40%, more preferably at least 50%, more preferablyat least 60%, more preferably at least 70%, more preferably at least80%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 100% of the DNA methyltransferaseactivity of the polypeptide comprising or consisting of the amino acidsequence shown as amino acids 1 to 337 of SEQ ID NO: 2. Bli1904IIrestriction endonuclease: The term “Bli1904II restriction endonuclease”is defined herein as a Type II restriction endonuclease that catalyzesthe site-specific endonucleolytic cleavage of DNA to give specificdouble-stranded DNA fragments (EC 3.1.21.4). For purposes of the presentinvention, Bli1904II restriction endonuclease activity is determinedaccording to established procedures for Type 11 restrictionendonucleases (e.g., Jeltsch and Pingoud, 2001, Methods Mol. Biol. 160:287-308). By definition, one unit of restriction endonuclease activitywill completely digest one μg of substrate DNA in a 50 μl reaction in 60minutes at 37° C.

The restriction endonucleases of the present invention have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the restrictionendonuclease activity of the polypeptide comprising or consisting of theamino acid sequence shown as amino acids 1 to 381 of SEQ ID NO: 4.

Restriction-modification system: The term “restriction-modificationsystem” is defined herein as a restriction endonuclease, a correspondingDNA methyltransferase that protects DNA from cleavage by the restrictionendonuclease, and the genes encoding these two enzymes.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2 or SEQ ID NO: 4, or ahomologous sequence thereof, wherein the fragment has DNAmethyltransferase or restriction endonuclease activity, respectively. Ina preferred aspect, a fragment of SEQ ID NO: 4 or a homolog thereofcontains at least 320 amino acid residues, more preferably at least 340amino acid residues, and most preferably at least 360 amino acidresidues. In another preferred aspect, a fragment of SEQ ID NO: 2 or ahomolog thereof contains at least 290 amino acid residues, morepreferably at least 305 amino acid residues, and most preferably atleast 320 amino acid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1 or SEQ ID NO: 3, or a homologous sequence thereof,wherein the subsequence encodes a polypeptide fragment having DNAmethyltransferase activity or restriction endonuclease, respectively. Ina preferred aspect, a subsequence of SEQ ID NO: 3 or a homolog thereofcontains at least 960 nucleotides, more preferably at least 1020nucleotides, and most preferably at least 1080 nucleotides. In anotherpreferred aspect, a subsequence of SEQ ID NO: 1 or a homolog thereofcontains at least 870 nucleotides, more preferably at least 915nucleotides, and most preferably at least 960 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or is modified tocontain segments of nucleic acids in a manner that would not otherwiseexist in nature or is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: he term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction,conjugation, and the like with a nucleic acid construct or expressionvector.

Introduction: The term “introduction” and variations thereof are definedherein as the transfer of a DNA into a bacterial cell. The introductionof a DNA into a bacterial cell can be accomplished by any method knownin the art, including, the not limited to, transformation, transfection,transduction, conjugation, and the like.

Transformation: The term “transformation” is defined herein asintroducing a purified DNA into a bacterial cell so that the DNA ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. The term “transformation” shall be generallyunderstood to include transfection, transduction, conjugation, and thelike.

Transfection: The term “transfection” is defined herein as thetransformation of a bacterial cell with a viral nucleic acid.

Transduction: The term “transduction” is defined herein as the packagingof DNA from a first bacterial cell into a virus particle and thetransfer of that bacterial DNA to a second bacterial cell by infectionof the second cell with the virus particle.

Conjugation: The term “conjugation” is defined herein as the transfer ofDNA directly from one bacterial cell to another bacterial cell throughcell-to-cell contact.

Transformant: The term “transformant” is defined herein to generallyencompass any bacterial host cell into which a DNA has been introduced.Consequently, the term “transformant” included transfectants,conjugants, and the like.

Donor Cell: The term “donor cell” is defined herein as a cell that isthe source of DNA introduced by any means to another cell.

Recipient cell: The term “recipient cell” is defined herein as a cellinto which DNA is introduced.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of amino acids 1 to 337 ofSEQ ID NO: 2 or amino acids 1 to 381 of SEQ ID NO: 4; or a homologoussequence thereof; as well as genetic manipulation of the DNA encodingsuch a polypeptide. The modification can be a substitution, a deletionand/or an insertion of one or more (several) amino acids as well asreplacements of one or more (several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having DNA methyltransferase or restrictionendonuclease activity produced by an organism expressing a modifiedpolynucleotide sequence of nucleotides 1 to 1011 of SEQ ID NO: 1 ornucleotides 1 to 1143 of SEQ ID NO: 3, respectively, or a homologoussequence thereof. The modified polynucleotide sequence is obtainedthrough human intervention by modification of the nucleotide sequencedisclosed in SEQ ID NO: 1 or SEQ ID NO: 3, or a homologous sequencethereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of producing bacterialtransformants, comprising: (a) introducing a DNA into a first bacterialhost cell comprising a polynucleotide encoding a DNA methyltransferaseto methylate the DNA; wherein the polynucleotide encoding the DNAmethyltransferase is selected from the group consisting of (i) apolynucleotide encoding a polypeptide comprising an amino acid sequencehaving at least 60% sequence identity with amino acids 1 to 337 of SEQID NO: 2; (ii) a polynucleotide comprising a nucleotide sequence havingat least 60% sequence identity with nucleotides 1 to 1011 of SEQ ID NO:1; (iii) a polynucleotide that hybridizes under at least mediumstringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or itsfull-length complementary strand; and (iv) a polynucleotide encoding avariant comprising a substitution, deletion, and/or insertion of one ormore amino acids of amino acids 1 to 337 of SEQ ID NO: 2; and whereinthe DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2; (b)transferring the methylated DNA from the first bacterial host cell intoa second bacterial host cell; wherein the second bacterial host cellcomprises a restriction endonuclease able to degrade the DNA but unableto degrade the methylated DNA; and (c) isolating transformants of thesecond bacterial host cell comprising the methylated DNA.

The present invention also relates to methods of producing bacterialtransformants, comprising: (a) methylating in vitro a DNA with a DNAmethyltransferase to produce a methylated DNA; wherein the DNAmethyltransferase is selected from the group consisting of (i) apolypeptide comprising an amino acid sequence having at least 60%sequence identity with amino acids 1 to 337 of SEQ ID NO: 2; (ii) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 60% sequence identity with nucleotides 1 to 1011 of SEQID NO: 1; (iii) a polypeptide encoded by a polynucleotide thathybridizes under at least medium stringency conditions with nucleotides1 to 1011 of SEQ ID NO: 1 or its full-length complementary strand; and(iv) a variant comprising a substitution, deletion, and/or insertion ofone or more amino acids of amino acids 1 to 337 of SEQ ID NO: 2; andwherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2; (b)introducing the methylated DNA into a bacterial host cell; wherein thebacterial host cell comprises a restriction endonuclease able to degradethe DNA but unable to degrade the methylated DNA; and (c) isolatingtransformants of the bacterial host cell comprising the methylated DNA

The present invention also relates to methods of producing bacterialtransformants, comprising: (a) introducing a DNA into a bacterial hostcell comprising a polynucleotide encoding a restriction endonuclease,which is inactivated; wherein the polynucleotide encoding therestriction endonuclease is selected from the group consisting of (i) apolynucleotide encoding a polypeptide comprising an amino acid sequencehaving at least 60% sequence identity with amino acids 1 to 381 of SEQID NO: 4; (ii) a polynucleotide comprising a nucleotide sequence havingat least 60% sequence identity with nucleotides 1 to 1143 of SEQ ID NO:3; (iii) a polynucleotide that hybridizes under at least mediumstringency conditions with nucleotides 1 to 1143 of SEQ ID NO: 3 or itsfull-length complementary strand; and (iv) a polynucleotide encoding avariant comprising a substitution, deletion, and/or insertion of one ormore amino acids of amino acids 1 to 381 of SEQ ID NO: 4; wherein therestriction endonuclease has the same specificity as the restrictionendonuclease of amino acids 1 to 381 of SEQ ID NO: 4, and wherein theinactivation of the polynucleotide encoding the restriction endonucleaseprevents the introduced DNA from being digested by the restrictionendonuclease and avoids the need to methylate the DNA with a DNAmethyltransferase prior to introducing the DNA into the bacterial hostcell, and (b) isolating transformants of the bacterial host cellcomprising the DNA.

In the methods of the present invention, the introduction of a DNA intoa bacterial host cell can be accomplished (1) by inactivating apolynucleotide encoding a restriction endonuclease of the bacterial hostcell so the introduced DNA is not degraded by the restrictionendonuclease or (2) by methylating the DNA with a DNA methyltransferaseprior to its introduction into a bacterial host cell, so the introducedmethylated DNA is not degraded by the restriction endonuclease of thebacterial host cell. The DNA methyltransferase or the restrictionendonuclease may be native or foreign to the bacterial host cell.

In one aspect, the methylation of the DNA can be accomplished in vitro.For example, a DNA methyltransferase of the present invention can berecovered and used to methylate the DNA in the presence ofS-adenosyl-L-methionine resulting in S-adenosyl-L-homocysteine and DNAcontaining 5-methylcytosine.

In another aspect, the methylation of the DNA can be accomplished invivo. For example, the methylation of the DNA can be accomplished bycloning the polynucleotide encoding the DNA methyltransferase from thebacterial host cell (recipient cell) and expressing the DNAmethyltransferase coding sequence in another bacterial cell (donor cell)where the DNA is introduced and methylated and then the methylated DNAis transferred or introduced into the bacterial host cell (recipientcell) from which the DNA methyltransferase coding sequence was isolated.In a further aspect, the polynucleotide encoding the DNAmethyltransferase may be obtained from another organism that is not therecipient cell. In an even further aspect, the donor host cell mayalready contain a polynucleotide encoding a DNA methyltransferase or ahomolog thereof of the present invention.

Introduction of the methylated DNA into the bacterial host cell can beaccomplished by transfer from the first bacterial cell into the secondbacterial cell, i.e., by conjugation, or by isolating the methylated DNAfrom the first bacterial cell and then introducing the isolatedmethylated DNA into the second bacterial cell by, for example,transformation.

The methods of the present invention increase the number oftransformants obtained by at least 10-fold, preferably at least100-fold, more preferably at least 1000-fold, even more preferably atleast 10,000-fold, and most preferably at least 100,000-fold compared toconventional methods without inactivating a restriction endonuclease ofthe present invention or methylating a DNA with a DNA methyltransferaseof the present invention.

Restriction Endonuclease Genes and DNA Methyltransferase Genes andEnzymes Thereof

In a first aspect, the present invention relates to isolatedpolynucleotides encoding polypeptides having DNA methyltransferaseactivity comprising an amino acid sequence having a degree of sequenceidentity to amino acids 1 to 337 of SEQ ID NO: 2 of preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which have DNA methyltransferase activity(hereinafter “homologous polypeptides” or “homologs”). In a preferredaspect, the homologous polypeptides having DNA methyltransferaseactivity comprise an amino acid sequence that differs by ten aminoacids, preferably by five amino acids, more preferably by four aminoacids, even more preferably by three amino acids, most preferably by twoamino acids, and even most preferably by one amino acid from amino acids1 to 337 of SEQ ID NO: 2.

An isolated polynucleotide of the present invention preferably encodes apolypeptide having DNA methyltransferase activity comprising the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has DNA methyltransferase activity. In a preferredaspect, a polypeptide comprises the amino acid sequence of SEQ ID NO: 2.In another preferred aspect, a polypeptide comprises amino acids 1 to337 of SEQ ID NO: 2, or an allelic variant thereof; or a fragmentthereof that has DNA methyltransferase activity. In another preferredaspect, a polypeptide comprises amino acids 1 to 337 of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has DNA methyltransferase activity. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:2. In another preferred aspect, a polypeptide consists of amino acids 1to 337 of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has DNA methyltransferase activity. In another preferredaspect, a polypeptide consists of amino acids 1 to 337 of SEQ ID NO: 2.

The present invention also relates to isolated polynucleotidescomprising nucleotide sequences that encode polypeptides having DNAmethyltransferase activity. In a preferred aspect, the nucleotidesequence is set forth in SEQ ID NO: 1. In another more preferred aspect,the nucleotide sequence is the sequence contained in plasmid pMDT138that is contained in Escherichia coli NRRL B-41967. In another preferredaspect, the nucleotide sequence is nucleotides 1 to 1011 of SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence isnucleotides 1 to 1011 of SEQ ID NO: 1 contained in plasmid pMDT138 thatis contained in Escherichia coli NRRL B-41967. The present inventionalso encompasses nucleotide sequences that encode a polypeptide havingthe amino acid sequence of SEQ ID NO: 2, which differ from SEQ ID NO: 1by virtue of the degeneracy of the genetic code. The present inventionalso relates to subsequences of SEQ ID NO: 1 that encode fragments ofSEQ ID NO: 2 that have methyltransferase activity.

In another first aspect, the present invention relates to isolatedpolynucleotides encoding polypeptides having restriction endonucleaseactivity comprising an amino acid sequence having a degree of sequenceidentity to amino acids 1 to 381 of SEQ ID NO: 4 of preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%, which have restriction endonuclease activity(hereinafter “homologous polypeptides” or “homologs”). In a preferredaspect, the homologous polypeptides having restriction endonucleaseactivity comprise an amino acid sequence that differs by ten aminoacids, preferably by five amino acids, more preferably by four aminoacids, even more preferably by three amino acids, most preferably by twoamino acids, and even most preferably by one amino acid from amino acids1 to 381 of SEQ ID NO: 4.

An isolated polynucleotide of the present invention preferably encodes apolypeptide having restriction endonuclease activity comprising theamino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof that has restriction endonuclease activity. In apreferred aspect, a polypeptide comprises the amino acid sequence of SEQID NO: 4. In another preferred aspect, a polypeptide comprises aminoacids 1 to 381 of SEQ ID NO: 4, or an allelic variant thereof; or afragment thereof that has restriction endonuclease activity. In anotherpreferred aspect, a polypeptide comprises amino acids 1 to 381 of SEQ IDNO: 4. In another preferred aspect, a polypeptide consists of the aminoacid sequence of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof that has restriction endonuclease activity. In anotherpreferred aspect, a polypeptide consists of the amino acid sequence ofSEQ ID NO: 4. In another preferred aspect, a polypeptide consists ofamino acids 1 to 381 of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof that has restriction endonuclease activity. In anotherpreferred aspect, a polypeptide consists of amino acids 1 to 381 of SEQID NO: 4.

The present invention also relates to isolated polynucleotidescomprising nucleotide sequences that encode polypeptides havingrestriction endonuclease activity. In another preferred aspect, thenucleotide sequence is set forth in SEQ ID NO: 3. In another morepreferred aspect, the nucleotide sequence is the sequence contained inplasmid pMDT156 that is contained in Escherichia coli NRRL B-41968. Inanother preferred aspect, the nucleotide sequence is nucleotides 1 to1143 of SEQ ID NO: 3. In another more preferred aspect, the nucleotidesequence is nucleotides 1 to 1143 of SEQ ID NO: 3 contained in plasmidpMDT156 that is contained in Escherichia coli NRRL B-41968. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 4, which differfrom SEQ ID NO: 3 by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 3 thatencode fragments of SEQ ID NO: 4 that have restriction endonucleaseactivity.

In a second aspect, the present invention relates to isolatedpolynucleotides encoding polypeptides having DNA methyltransferaseactivity that hybridize under very low stringency conditions, preferablylow stringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with (i) nucleotides 1 to 1011 of SEQ ID NO: 1, (ii) asubsequence of (i), or (iii) a full-length complementary strand of (i)or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y. Asubsequence of SEQ ID NO: 3 contains at least 100 contiguous nucleotidesor preferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment that has DNAmethyltransferase activity.

In another second aspect, the present invention relates to isolatedpolynucleotides encoding polypeptides having restriction endonucleaseactivity that hybridize under very low stringency conditions, preferablylow stringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with (i) nucleotides 1 to 1143 of SEQ ID NO: 3, (ii) asubsequence of (i), or (iii) a full-length complementary strand of (i)or (ii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, supra). Asubsequence of SEQ ID NO: 3 contains at least 100 contiguous nucleotidesor preferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment that has restrictionendonuclease activity.

The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4, or a fragment thereof, may be used to design a nucleicacid probe to identify and clone DNA encoding polypeptides havingrestriction endonuclease activity or DNA methyltransferase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes that are at least 600 nucleotides, atleast preferably at least 700 nucleotides, more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used.

The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA library prepared from such other organisms may, therefore,be screened for DNA that hybridizes with the probes described above andencodes a polypeptide having restriction endonuclease activity or DNAmethyltransferase activity. Genomic DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1 or SEQ ID NO: 3, or a subsequence thereof, the carriermaterial is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ IDNO: 3, its full-length complementary strand, or a subsequence thereof,under very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridizes under these conditions can be detectedusing X-ray film.

In a preferred aspect, the nucleic acid probe is a polynucleotidecomprising a nucleotide sequence that encodes the polypeptide of SEQ IDNO: 2, or a subsequence thereof. In another preferred aspect, thenucleic acid probe is SEQ ID NO: 1 or its full-length complementarystrand. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region of SEQ ID NO: 1. In another preferredaspect, the nucleic acid probe is the polynucleotide contained inplasmid pMDT138 which is contained in Escherichia coli NRRL B-41967,wherein the nucleotide sequence thereof encodes a polypeptide having DNAmethyltransferase activity. In another preferred aspect, the nucleicacid probe is the mature polypeptide coding region contained in plasmidpMDT138 which is contained in Escherichia coli NRRL B-41967.

In another preferred aspect, the nucleic acid probe is a polynucleotidecomprising a nucleotide sequence that encodes the polypeptide of SEQ IDNO: 4, or a subsequence thereof. In another preferred aspect, thenucleic acid probe is SEQ ID NO: 3 or its full-length complementarystrand. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is the polynucleotide contained inplasmid pMDT156 which is contained in Escherichia coli NRRL B-41968,wherein the nucleotide sequence thereof encodes a polypeptide havingrestriction endonuclease activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding region contained inplasmid pMDT156 which is contained in Escherichia coli NRRL B-41968.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(n), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolynucleotides encoding artificial variants comprising a substitution,deletion, and/or insertion of one or more amino acids of SEQ ID NO: 2 orSEQ ID NO: 4, or a homologous sequence thereof; or the maturepolypeptide thereof. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions or insertions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic

Press, New York. The most commonly occurring exchanges are Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, andAsp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,restriction endonuclease or DNA methyltransferase activity) to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to a polypeptide according to theinvention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of amino acids 1 to 337 of SEQ ID NO: 2 is 10, preferably 9,more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of amino acids 1 to 381 of SEQ ID NO: 4 is 10, preferably 9,more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

The present invention also relates to isolated DNA methyltransferasesselected from the group consisting of (a) a polypeptide comprising anamino acid sequence having preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with amino acids 1 to 337 of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith nucleotides 1 to 1011 of SEQ ID NO: 1; (c) a polypeptide encoded bya polynucleotide that hybridizes under preferably at least mediumstringency conditions, more preferably at least medium-high stringencyconditions, and most preferably at least high stringency conditions withnucleotides 1 to 1011 of SEQ ID NO: 1 or its full-length complementarystrand; and (d) a variant comprising a substitution, deletion, and/orinsertion of one or more amino acids of amino acids 1 to 337 of SEQ IDNO: 2.

The present invention also relates to isolated restriction endonucleasesselected from the group consisting of (a) a polypeptide comprising anamino acid sequence having preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with amino acids 1 to 381 of SEQ ID NO: 4; (b) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith nucleotides 1 to 1143 of SEQ ID NO: 3; (c) a polypeptide encoded bya polynucleotide that hybridizes under preferably at least mediumstringency conditions, more preferably at least medium-high stringencyconditions, and most preferably at least high stringency conditions withnucleotides 1 to 1143 of SEQ ID NO: 3 or its full-length complementarystrand; and (d) a variant comprising a substitution, deletion, and/orinsertion of one or more amino acids of amino acids 1 to 381 of SEQ IDNO: 4.

Inactivation of Restriction Endonuclease Genes

In the methods of the present invention, the introduction of a DNA intoa bacterial host cell can be accomplished by inactivating a restrictionendonuclease encoding polynucleotide native or foreign to the bacterialhost cell so the introduced DNA is not degraded by the restrictionendonuclease. The polynucleotide may be a component of arestriction-modification system.

Inactivation of a restriction endonuclease gene or a homolog thereof ofthe present invention in a bacterial host cell may be accomplished usingmethods well known in the art, for example, insertions, disruptions,replacements, or deletions of the gene. The portion of the gene to beinactivated may be, for example, the coding region or a regulatoryelement required for expression of the coding region. An example of sucha regulatory or control sequence may be a promoter sequence or afunctional part thereof, i.e., a part that is sufficient for affectingexpression of the nucleotide sequence. Other control sequences forpossible modification include, but are not limited to, a leader,propeptide sequence, signal sequence, transcription terminator, andtranscriptional activator.

Inactivation of a restriction endonuclease gene of the present inventionmay be achieved by gene deletion techniques to eliminate or reduce theexpression of the gene. Gene deletion techniques enable the partial orcomplete removal of the gene thereby eliminating its expression. In suchmethods, the deletion of the gene may be accomplished by homologousrecombination using a plasmid that has been constructed to contiguouslycontain 5′ and 3′ regions flanking the gene. The contiguous 5′ and 3′regions may be introduced into a bacterial cell, for example, on atemperature-sensitive plasmid, such as pE194, in association with aselectable marker at a permissive temperature to allow the plasmid tobecome established in the cell. The cell is then shifted to anon-permissive temperature to select for cells that have the plasmidintegrated into the chromosome at one of the homologous flankingregions. Selection for integration of the plasmid is effected byselection for the selectable marker. After integration, a recombinationevent at the second homologous flanking region is stimulated by shiftingthe cells to the permissive temperature for several generations withoutselection. The cells are plated to obtain single colonies and thecolonies are examined for loss of the selectable marker (see, forexample, Perego, 1993, In A. L. Sonneshein, J. A. Hoch, and R. Losick,editors, Bacillus subtilis and Other Gram-Positive Bacteria, Chapter 42,American Society of Microbiology, Washington, D.C.).

Inactivation of a restriction endonuclease gene of the present inventionmay also be accomplished by introducing, substituting, or removing oneor more nucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a frame-shift of the open readingframe. Such a modification may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. See, for example, Botstein and Shortie, 1985, Science229: 1193-1201; Lo et al., 1984, Proceedings of the National Academy ofSciences USA 81: 2285-2289; Higuchi et al., 1988, Nucleic Acids Research16: 7351-7367; Shimada, 1996, Meth. Mol. Biol. 57: 157-165; Ho et al.,1989, Gene 77: 51-59; Horton et al., 1989, Gene 77: 61-68; and Sarkarand Sommer, 1990, BioTechniques 8: 404-407.

Inactivation of a restriction endonuclease gene of the present inventionmay also be accomplished by gene disruption techniques by inserting intothe gene an integrative plasmid containing a nucleic acid fragmenthomologous to the gene, which will create a duplication of the region ofhomology and incorporate vector DNA between the duplicated regions. Suchgene disruption can eliminate gene expression if the inserted vectorseparates the promoter of the gene from the coding region or interruptsthe coding sequence such that a non-functional gene product results. Adisrupting construct may be simply a selectable marker gene accompaniedby 5′ and 3′ regions homologous to the gene. The selectable markerenables identification of transformants containing the disrupted gene.

Inactivation of a restriction endonuclease gene of the present inventionmay also be accomplished by the process of gene conversion (see, forexample, Iglesias and Trautner, 1983, Molecular General Genetics 189:73-76). For example, in the gene conversion method, a nucleotidesequence corresponding to the gene is mutagenized in vitro to produce adefective nucleotide sequence that is then transformed into the parentbacterial cell to produce a defective gene. By homologous recombination,the defective nucleotide sequence replaces the gene. It may be desirablethat the defective gene or gene fragment also encodes a marker that maybe used for selection of transformants containing the defective gene.For example, the defective gene may be introduced on a non-replicatingor temperature-sensitive plasmid in association with a selectablemarker. Selection for integration of the plasmid is effected byselection for the marker under conditions not permitting plasmidreplication. Selection for a second recombination event leading to genereplacement is effected by examination of colonies for loss of theselectable marker and acquisition of the mutated gene (see, for example,Perego, 1993, supra). Alternatively, the defective nucleotide sequencemay contain an insertion, substitution, or deletion of one or morenucleotides of the gene, as described below.

Inactivation of a restriction endonuclease gene of the present inventionmay also be accomplished by established anti-sense techniques using anucleotide sequence complementary to the nucleotide sequence of the gene(Parish and Stoker, 1997, FEMS Microbiology Letters 154: 151-157). Morespecifically, expression of the gene by a bacterial cell may be reducedor eliminated by introducing a nucleotide sequence complementary to thenucleotide sequence of the gene, which may be transcribed in the celland is capable of hybridizing to the mRNA produced in the cell. Underconditions allowing the complementary anti-sense nucleotide sequence tohybridize to the mRNA, the amount of protein translated is thus reducedor eliminated. Inactivation may also be accomplished by RNA interferencetechniques (see, for example, U.S. Pat. No. 6,506,559).

Inactivation of a restriction endonuclease gene of the present inventionmay be further accomplished by random or specific mutagenesis usingmethods well known in the art, including, but not limited to, chemicalmutagenesis (see, for example, Hopwood, The Isolation of Mutants inMethods in Microbiology (J. R. Norris and D. W. Ribbons, eds.) pp363-433, Academic Press, New York, 1970) and transposition (see, forexample, Youngman et al., 1983, Proc. Natl. Acad. Sci. USA 80:2305-2309). Modification of the gene may be performed by subjecting theparent cell to mutagenesis and screening for mutant cells in whichexpression of the gene has been reduced or eliminated. The mutagenesis,which may be specific or random, may be performed, for example, by useof a suitable physical or chemical mutagenizing agent, use of a suitableoligonucleotide, or subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the mutagenesis may be performed by use of anycombination of these mutagenizing methods.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosoguanidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the parent cell to be mutagenized inthe presence of the mutagenizing agent of choice under suitableconditions, and selecting for mutant cells exhibiting reduced or noexpression of the gene.

In a preferred aspect, the inactivation of a restriction endonucleasegene in the bacterial host cell is unmarked with a selectable marker.

Removal of the selectable marker gene may be obtained by culturing themutants on a counter-selection medium. Where the selectable marker genecontains repeats flanking its 5′ and 3′ ends, the repeats willfacilitate the looping out of the selectable marker gene by homologousrecombination when the mutant cell is submitted to counter-selection.The selectable marker gene may also be removed by homologousrecombination by introducing into the mutant cell a nucleic acidfragment comprising 5′ and 3′ regions of the defective gene, but lackingthe selectable marker gene, followed by selecting on thecounter-selection medium. By homologous recombination, the defectivegene containing the selectable marker gene is replaced with the nucleicacid fragment lacking the selectable marker gene. Other methods known inthe art may also be used.

Nucleic Acid Constructs

A polynucleotide encoding a polypeptide of interest, e.g., a polypeptidehaving biological activity, or a DNA methyltransferase or a restrictionendonuclease of the present invention, can be manipulated in a varietyof ways to provide for expression of the polynucleotide in a suitablebacterial host cell, for example, to enable the methylation of a DNA ofinterest or for production and recovery of the DNA methyltransferase soit can be used in in vitro methylation. Manipulation of thepolynucleotide's nucleotide sequence prior to its insertion into anucleic acid construct or vector may be desirable or necessary dependingon the nucleic acid construct or vector or bacterial host cell. Thetechniques for modifying nucleotide sequences utilizing cloning methodsare well known in the art.

A nucleic acid construct comprising a polynucleotide encoding apolypeptide of interest may be operably linked to one or more controlsequences capable of directing the expression of the coding sequence ina bacterial host cell under conditions compatible with the controlsequences.

Each control sequence may be native or foreign to the nucleotidesequence encoding a polypeptide of interest. Such control sequencesinclude, but are not limited to, a leader, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleotidesequence.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a bacterial host cell forexpression of the nucleotide sequence. The promoter sequence containstranscription control sequences that mediate the expression of thepolypeptide of interest. The promoter may be any nucleotide sequencethat shows transcriptional activity in the bacterial host cell of choiceand may be obtained from genes directing synthesis of extracellular orintracellular polypeptides having biological activity either homologousor heterologous to the bacterial host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a bacterial cell arethe promoters obtained from the E. coli lac operon, the Streptomycescoelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene(sacB), the Bacillus licheniformis alpha-amylase gene (amyL), theBacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillusamyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformispenicillinase gene (penP), the Bacillus subtilis xylA and xylB genes,and the prokaryotic beta-lactamase gene (VIIIa-Komaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75:3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80:21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242:74-94; and in J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a bacterial cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding a DNA methyltransferase.Any terminator that is functional in the bacterial host cell of choicemay be used in the present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA that is important for translation by thebacterial cell. The leader sequence is operably linked to the 5′terminus of the nucleotide sequence directing synthesis of thepolypeptide having biological activity. Any leader sequence that isfunctional in the bacterial host cell of choice may be used in thepresent invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of apolypeptide that can direct the expressed polypeptide into the cell'ssecretory pathway. The signal peptide coding region may be native to thepolypeptide or may be obtained from foreign sources. The 5′ end of thecoding sequence of the nucleotide sequence may inherently contain asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding region that is foreign to that portionof the coding sequence and encodes the secreted polypeptide. The foreignsignal peptide coding region may be required where the coding sequencedoes not normally contain a signal peptide coding region. Alternatively,the foreign signal peptide coding region may simply replace the naturalsignal peptide coding region in order to obtain enhanced secretion ofthe polypeptide relative to the natural signal peptide coding regionnormally associated with the coding sequence. The signal peptide codingregion may be obtained from an amylase or a protease gene from aBacillus species. However, any signal peptide coding region capable ofdirecting the expressed polypeptide into the secretory pathway of abacterial host cell of choice may be used in the present invention.

An effective signal peptide coding region for bacterial cells, e.g.,Bacillus cells, is the signal peptide coding region obtained from themaltogenic amylase gene from Bacillus NCIB 11837, the Bacillusstearothermophilus alpha-amylase gene, the Bacillus licheniformissubtilisin gene, the Bacillus licheniformis beta-lactamase gene, theBacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM),and the Bacillus subtilis prsA gene. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews57:109-137.

The control sequence may also be a mRNA stabilizing sequence. The term“mRNA stabilizing sequence” is defined herein as a sequence locateddownstream of a promoter region and upstream of a coding sequence of apolynucleotide to which the promoter region is operably linked such thatall mRNAs synthesized from the promoter region may be processed togenerate mRNA transcripts with a stabilizer sequence at the 5′ end ofthe transcripts. The presence of such a stabilizer sequence at the 5′end of the mRNA transcripts increases their half-life (Agaisse andLereclus, 1994, supra, Hue et al., 1995, Journal of Bacteriology 177:3465-3471). The mRNA processing/stabilizing sequence is complementary tothe 3′ extremity of bacterial 16S ribosomal RNA. In a preferred aspect,the mRNA processing/stabilizing sequence generates essentiallysingle-size transcripts with a stabilizing sequence at the 5′ end of thetranscripts. The mRNA processing/stabilizing sequence is preferably one,which is complementary to the 3′ extremity of a bacterial 16S ribosomalRNA. See, U.S. Pat. Nos. 6,255,076 and 5,955,310.

An effective mRNA processing/stabilizing sequence for bacterial cells isthe Bacillus thuringiensis cryIIIA mRNA processing/stabilizing sequencedisclosed in WO 94/25612, or portions thereof, which retain the mRNAprocessing/stabilizing function, or the Bacillus subtilis SP82 mRNAprocessing/stabilizing sequence disclosed in Hue et al., 1995, Journalof Bacteriology 177: 3465-3471, or portions thereof, which retain themRNA processing/stabilizing function.

The nucleic acid construct can then be introduced into a bacterial hostcell using methods known in the art or those methods described hereinfor expressing the polypeptide of interest.

Recombinant Expression Vectors

In the methods of the present invention, a recombinant expression vectorcomprising a polynucleotide encoding a polypeptide of interest, e.g., apolypeptide having biological activity, or a DNA methyltransferase or arestriction endonuclease of the present invention, a promoter, andtranscriptional and translational stop signals may be used for therecombinant production of the polypeptide. The various nucleic acid andcontrol sequences described above may be joined together to produce arecombinant expression vector that may include one or more convenientrestriction sites to allow for insertion or substitution of thenucleotide sequence directing synthesis of a polypeptide of interest atsuch sites. Alternatively, the nucleotide sequence may be expressed byinserting the nucleotide sequence or a nucleic acid construct comprisingthe sequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression, and possibly secretion.

The recombinant expression vector may be any vector that can beconveniently subjected to recombinant DNA procedures and can bring aboutthe expression of the nucleotide sequence. The choice of the vector willtypically depend on the compatibility of the vector with the bacterialcell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone that, when introduced into the bacterial cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The vector system may be a single vector or plasmidor two or more vectors or plasmids that together contain the total DNAto be introduced into the genome of the Bacillus cell, or a transposon.

The vectors may be integrated into the bacterial cell genome whenintroduced into a bacterial cell. For integration into the host cellgenome, the vector may rely on the polynucleotide's sequence encodingthe polypeptide of interest or any other element of the vector forintegration into the genome by homologous or nonhomologousrecombination. Alternatively, the vector may contain additionalnucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of sequence identity to thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleotide sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in thebacterial cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pBR322, pUC19, pACYC177, andpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,and pAMβ1 permitting replication in Bacillus. The origin of replicationmay be one having a mutation to make its function temperature-sensitivein the bacterial cell (see, e.g., Ehrlich, 1978, Proceedings of theNational Academy of Sciences USA 75:1433-1436).

More than one copy of a nucleotide sequence directing synthesis of apolypeptide of interest may be introduced into the bacterial cell toamplify expression of the nucleotide sequence. Stable amplification ofthe nucleotide sequence can be obtained by integrating at least oneadditional copy of the sequence into the bacterial cell genome usingmethods well known in the art and selecting for transformants. Aconvenient method for achieving amplification of genomic DNA sequencesis described in WO 94/14968.

The vectors preferably contain one or more selectable markers thatpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers that confer antibiotic resistancesuch as ampicillin, kanamycin, erythromycin, chloramphenicol ortetracycline resistance. Furthermore, selection may be accomplished byco-transformation, e.g., as described in WO 91/09129, where theselectable marker is on a separate vector.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98:6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the for introducing DNA into a host cell can be used.

Bacterial Host Cells

The present invention also relates to bacterial host cells comprising anucleic acid construct or recombinant expression vector comprising apolynucleotide encoding a polypeptide of interest, e.g., a polypeptidehaving biological activity, or a restriction endonuclease or DNAmethyltransferase of the present invention.

The present invention also relates to bacterial host cells comprising apolynucleotide encoding a restriction endonuclease, which isinactivated;

wherein the polynucleotide encoding the restriction endonuclease isselected from the group consisting of (a) a polynucleotide encoding apolypeptide comprising an amino acid sequence having preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99% sequence identity with amino acids 1 to 381 of SEQID NO: 4; (b) a polynucleotide comprising a nucleotide sequence havingpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity withnucleotides 1 to 1143 of SEQ ID NO: 3; and (c) a polynucleotide thathybridizes under preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, and mostpreferably at least high stringency conditions with nucleotides 1 to1143 of SEQ ID NO: 3 or its full-length complementary strand;

wherein the restriction endonuclease has the same specificity as therestriction endonuclease of amino acids 1 to 381 of SEQ ID NO: 4; and

wherein the inactivation of the polynucleotide encoding the restrictionendonuclease prevents DNA that is introduced into the bacterial hostcell from being digested by the restriction endonuclease and avoids theneed to methylate the DNA with a DNA methyltransferase prior to beingintroduced into the host cell.

The present invention also relates to bacterial host cells comprising anucleic acid construct or recombinant expression vector comprising a DNAencoding or involved in the expression of a polypeptide havingbiological activity, wherein the bacterial host cells comprising the DNAis obtained by one of the methods described herein for introducing theDNA into such host cells.

The bacterial host cell may be any Gram positive bacterium or a Gramnegative bacterium that comprises a restriction-modification system, asdescribed herein. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

In the methods of the present invention, the bacterial host cell may beany Bacillus cell. Bacillus cells useful in the practice of the presentinvention include, but are not limited to, Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

In the methods of the present invention, the bacterial host cell may beany Streptococcus cell. Streptococcus cells useful in the practice ofthe present invention include, but are not limited to, Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, andStreptococcus equi subsp. Zooepidemicus.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

In the methods of the present invention, the bacterial host cell may beany Streptomyces cell. Streptomyces cells useful in the practice of thepresent invention include, but are not limited to, Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, and Streptomyces lividans.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

In the methods of the present invention involving methylation of a DNAof interest with a DNA methyltransferase, the bacterial cell used forthe methylation will likely be different from the final host bacterialcell. The bacterial cell used for the methylation can be a bacterialcell into which a DNA methyltransferase gene of the present invention,foreign to the cell, has been introduced or a bacterial cell in which aDNA methyltransferase gene is native to the cell.

In a further aspect of the present invention, the bacterial host cellmay additionally contain modifications, e.g., deletions or disruptions,of other genes that may be detrimental to the production, recovery orapplication of a polypeptide of interest. In a preferred aspect, abacterial host cell is a protease-deficient cell. In a more preferredaspect, the bacterial host cell, e.g., Bacillus cell, comprises adisruption or deletion of aprE and nprE. In another preferred aspect,the bacterial host cell does not produce spores. In another morepreferred aspect, the bacterial host cell, e.g., Bacillus cell,comprises a disruption or deletion of spoIIAC. In another preferredaspect, the bacterial host cell, e.g., Bacillus cell, comprises adisruption or deletion of one of the genes involved in the biosynthesisof surfactin, e.g., srfA, srfB, srfC, and srfD. See, for example, U.S.Pat. No. 5,958,728. Other genes, e.g., the amyE gene, which aredetrimental to the production, recovery or application of a polypeptideof interest may also be disrupted or deleted.

DNA

In the methods of the present invention, the DNA introduced into abacterial host cell can be any DNA of interest. The DNA may be native orheterologous (foreign) to the bacterial host cell of interest.

The DNA can encode any polypeptide having a biological activity ofinterest. The polypeptide may be native or heterologous (foreign) to thebacterial host cell of interest. The term “heterologous polypeptide” isdefined herein as a polypeptide that is not native to the host cell; anative polypeptide in which structural modifications, e.g., deletions,substitutions, and/or insertions, have been made to alter the nativepolypeptide; or a native polypeptide whose expression is quantitativelyaltered as a result of manipulation of the DNA encoding the polypeptideby recombinant DNA techniques, e.g., a stronger promoter. Thepolypeptide may be a naturally occurring allelic and engineeredvariations of the below-mentioned polypeptides and hybrid polypeptides.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins. The term “polypeptide” also encompasseshybrid polypeptides, which comprise a combination of partial or completepolypeptide sequences obtained from at least two different polypeptideswherein one or more may be heterologous to the fungal cell.

Polypeptides further include naturally occurring allelic and engineeredvariations of a polypeptide.

In a preferred aspect, the polypeptide is an antibody, antigen,antimicrobial peptide, enzyme, growth factor, hormone, immunodilator,neurotransmitter, receptor, reporter protein, structural protein, andtranscription factor.

In a more preferred aspect, the polypeptide is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In a most preferredaspect, the polypeptide is an alpha-glucosidase, aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase,laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme,peroxidase, phospholipase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, urokinase, or xylanase.

In another preferred aspect, the polypeptide is an albumin, collagen,tropoelastin, elastin, or gelatin.

In another preferred aspect, the polypeptide is a hybrid polypeptide,which comprises a combination of partial or complete polypeptidesequences obtained from at least two different polypeptides wherein oneor more may be heterologous to the bacterial host cell.

In another preferred aspect, the polypeptide is a fused polypeptide inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleotide sequence (or a portion thereof) encoding onepolypeptide to a nucleotide sequence (or a portion thereof) encodinganother polypeptide. Techniques for producing fusion polypeptides areknown in the art, and include, ligating the coding sequences encodingthe polypeptides so that they are in frame and expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

The DNA encoding a polypeptide of interest may be obtained from anyprokaryotic, eukaryotic, or other source. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide is produced by the sourceor by a cell in which a gene from the source has been inserted.

The techniques used to isolate or clone a DNA encoding a polypeptide ofinterest are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of the DNAof interest from such genomic DNA can be effected, e.g., by using thewell known polymerase chain reaction (PCR). See, for example, Innis etal., 1990, PCR Protocols: A Guide to Methods and Application, AcademicPress, New York. The cloning procedures may involve excision andisolation of a desired nucleic acid fragment comprising the nucleic acidsequence encoding the polypeptide, insertion of the fragment into avector molecule, and incorporation of the recombinant vector into themutant fungal cell where multiple copies or clones of the nucleic acidsequence will be replicated. The DNA may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

A DNA encoding a polypeptide of interest may be manipulated in a varietyof ways to provide for expression of the DNA in a suitable bacterialhost cell. The construction of nucleic acid constructs and recombinantexpression vectors for the DNA encoding a polypeptide of interest can becarried out as described herein for the expression of a DNAmethyltransferase of the present invention.

The DNA can also be a control sequence, e.g., promoter, for manipulatingthe expression of a gene of interest. Non-limiting examples of controlsequences are described herein.

The DNA can further be a nucleic acid construct for inactivating a geneof interest in a bacterial cell.

The DNA is not to be limited in scope by the specific examples disclosedabove, since these examples are intended as illustrations of severalaspects of the invention.

Methods of Production

The present invention also relates to methods of producing a polypeptidehaving biological activity, comprising:

(a) cultivating a bacterial host cell comprising an introduced DNAencoding or involved in the expression of the polypeptide havingbiological activity under conditions conducive for production of thepolypeptide;

wherein the DNA is methylated prior to being introduced into thebacterial host cell by a DNA methyltransferase selected from the groupconsisting of (i) a polypeptide comprising an amino acid sequence havingpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity with aminoacids 1 to 337 of SEQ ID NO: 2; (ii) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 60%, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99% sequence identity with nucleotides 1to 1011 of SEQ ID NO: 1; (iii) a polypeptide encoded by a polynucleotidethat hybridizes under preferably at least medium stringency conditions,more preferably at least medium-high stringency conditions, and mostpreferably at least high stringency conditions with nucleotides 1 to1011 of SEQ ID NO: 1 or its full-length complementary strand; and (iv) avariant comprising a substitution, deletion, and/or insertion of one ormore amino acids of amino acids 1 to 337 of SEQ ID NO: 2;

wherein the DNA methyltransferase has the same specificity as the DNAmethyltransferase of amino acids 1 to 337 of SEQ ID NO: 2 and is nativeto the bacterial host cell; and

wherein the methylation prevents the introduced DNA from being digestedby a restriction endonuclease of the bacterial host cell; and

(b) recovering the polypeptide having biological activity.

The present invention also relates to methods of producing a polypeptidehaving biological activity, comprising:

(a) cultivating a bacterial host cell comprising an introduced DNAencoding or involved in the expression of the polypeptide havingbiological activity under conditions conducive for production of thepolypeptide;

wherein the bacterial host cell comprises a polynucleotide encoding arestriction endonuclease, which is inactivated;

wherein the polynucleotide encoding the restriction endonuclease isselected from the group consisting of (i) a polynucleotide encoding apolypeptide comprising an amino acid sequence having preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99% sequence identity with amino acids 1 to 381 of SEQID NO: 4; (ii) a polynucleotide comprising a nucleotide sequence havingpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity withnucleotides 1 to 1143 of SEQ ID NO: 3; and (iii) a polynucleotide thathybridizes under preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, and mostpreferably at least high stringency conditions with nucleotides 1 to1143 of SEQ ID NO: 3 or its full-length complementary strand;

wherein the restriction endonuclease has the same specificity as therestriction endonuclease of amino acids 1 to 381 of SEQ ID NO: 4; and

wherein the inactivation of the polynucleotide encoding the restrictionendonuclease prevents the introduced DNA from being digested by therestriction endonuclease and avoids the need to methylate the DNA with aDNA methyltransferase prior to introducing the DNA into the bacterialhost cell; and

(b) recovering the polypeptide having biological activity.

The present invention also relates to methods of producing a polypeptidehaving restriction endonuclease activity, comprising: (a) cultivating abacterial host cell comprising a polynucleotide encoding a polypeptidehaving restriction endonuclease activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide having restriction endonuclease activity.

The present invention also relates to methods of producing a polypeptidehaving DNA methyltransferase activity, comprising: (a) cultivating abacterial host cell comprising a polynucleotide encoding a polypeptidehaving DNA methyltransferase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide having DNA methyltransferase activity.

The bacterial host cells are cultivated in a nutrient medium suitablefor production of a polypeptide of interest using methods known in theart. For example, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide of interest to be expressed and/or isolated. Thecultivation takes place in a suitable nutrient medium comprising carbonand nitrogen sources and inorganic salts, using procedures known in theart. Suitable media are available from commercial suppliers or may beprepared according to published compositions (e.g., in catalogues of theAmerican Type Culture Collection). The secreted polypeptide of interestcan be recovered directly from the medium.

The polypeptide of interest may be detected using methods known in theart that are specific for the polypeptide. These detection methods mayinclude use of specific antibodies, high performance liquidchromatography, capillary chromatography, formation of an enzymeproduct, disappearance of an enzyme substrate, or SDS-PAGE.

For example, an enzyme assay may be used to determine the activity ofthe enzyme. Procedures for determining enzyme activity are known in theart for many enzymes (see, for example, D. Schomburg and M. Salzmann(eds.), Enzyme Handbook, Springer-Verlag, New York, 1990). Assays fordetermining activity of a restriction endonuclease or DNAmethyltransferase of the present invention are described herein.

The resulting polypeptide may be isolated by methods known in the art.For example, a polypeptide of interest may be isolated from thecultivation medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation. The isolated polypeptide may then be further purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Inactivation of Genes

The present invention also relates to methods of producing a mutant of aparent bacterial cell, which comprises (a) introducing into a parentbacterial cell a DNA comprising a nucleic acid construct to inactivate agene encoding a polypeptide in the parent bacterial cell, which resultsin a mutant cell producing less of the polypeptide than the parent cellwhen cultivated under the same conditions; wherein the bacterial hostcell comprises a polynucleotide encoding a restriction endonuclease,which is inactivated, and the polynucleotide encoding the restrictionendonuclease is selected from the group consisting of (i) apolynucleotide encoding a polypeptide comprising an amino acid sequencehaving preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99% sequence identitywith amino acids 1 to 381 of SEQ ID NO: 4; (ii) a polynucleotidecomprising a nucleotide sequence having preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity with nucleotides 1 to 1143 of SEQ ID NO: 3;(iii) a polynucleotide that hybridizes under preferably at least mediumstringency conditions, more preferably at least medium-high stringencyconditions, and most preferably at least high stringency conditions withnucleotides 1 to 1143 of SEQ ID NO: 3 or its full-length complementarystrand; and (iv) a polynucleotide encoding a variant comprising asubstitution, deletion, and/or insertion of one or more amino acids ofamino acids 1 to 381 of SEQ ID NO: 4; wherein the restrictionendonuclease has the same specificity as the restriction endonuclease ofamino acids 1 to 381 of SEQ ID NO: 4, and wherein the inactivation ofthe polynucleotide encoding the restriction endonuclease prevents theintroduced DNA from being digested by the restriction endonuclease andavoids the need to methylate the DNA with a DNA methyltransferase priorto introducing the DNA into the parent bacterial cell; and (b) isolatingthe mutant cell.

The present invention also relates to methods of producing a mutant of aparent bacterial cell, which comprises (a) introducing into a parentbacterial cell a DNA comprising a nucleic acid construct to inactivate agene encoding a polypeptide in the parent bacterial cell, which resultsin a mutant cell producing less of the polypeptide than the parent cellwhen cultivated under the same conditions; wherein the DNA is methylatedprior to being introduced into the bacterial host cell by a DNAmethyltransferase selected from the group consisting of (i) apolypeptide comprising an amino acid sequence having preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, or at least 99%% sequence identity with amino acids 1 to 337 of SEQID NO: 2; (ii) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having preferably at least 60%, more preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1; (iii) apolypeptide encoded by a polynucleotide that hybridizes under preferablyat least medium stringency conditions, more preferably at leastmedium-high stringency conditions, and most preferably at least highstringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 or itsfull-length complementary strand; and (iv) a variant comprising asubstitution, deletion, and/or insertion of one or more amino acids ofamino acids 1 to 337 of SEQ ID NO: 2; wherein the DNA methyltransferasehas the same specificity as the DNA methyltransferase of amino acids 1to 337 of SEQ ID NO: 2; and wherein the methylation prevents theintroduced DNA from being digested by a restriction endonuclease of theparent bacterial cell; and (b) isolating the mutant cell.

The mutant cell comprising an inactivated gene may be constructed usingthe methods described here.

The bacterial mutant cells so created are particularly useful as hostcells for the expression of polypeptides native or foreign to the cells.Therefore, the present invention further relates to methods of producinga native or foreign polypeptide comprising: (a) cultivating the mutantcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide. The term “foreign polypeptide” isdefined herein as a polypeptide that is not native to the host cell, anative protein in which modifications have been made to alter the nativesequence, or a native protein whose expression is quantitatively alteredas a result of a manipulation of the host cell by recombinant DNAtechniques.

Examples of polypeptides that can be expressed in such mutants aredescribed herein.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art and describedherein.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES DNA Sequencing

DNA sequencing was performed using an Applied Biosystems Model 3130XGenetic Analyzer (Applied Biosystems, Foster City, Calif., USA)employing dye terminator chemistry (Giesecke et al., 1992, Journal ofVirol. Methods 38: 47-60). Sequences were assembled usingphred/phrap/consed (University of Washington, Seattle, Wash., USA) withsequence specific primers.

Strains

Bacillus plasmids were constructed in Bacillus subtilis 168Δ4. Bacillussubtilis 168Δ4 is derived from the Bacillus subtilis type strain 168(BGSC 1A1, Bacillus Genetic Stock Center, Columbus, Ohio) and hasdeletions in the spoIIAC, aprE, nprE, and amyE genes. The deletion ofthese four genes was performed essentially as described for Bacillussubtilis A164Δ5, which is described in detail in U.S. Pat. No.5,891,701.

Media

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl.

LB plates were composed of LB medium and 15 g of bacto agar per liter.

LB ampicillin medium was composed of LB medium and 100 μg of ampicillinper ml (filter sterilized, added after autoclaving).

LB ampicillin plates were composed of LB ampicillin medium and 15 g ofbacto agar per liter.

VY medium was composed per liter of 25 g of veal infusion (BDDiagnostics, Franklin Lakes, N.J., USA) and 5 g of yeast extract.

2×YT medium was composed per liter of 16 g of Tryptone, 10 g of yeastextract, and 5 g of NaCl.

2×YT ampicillin medium was composed of 2×YT medium and 100 μg ofampicillin per ml (filter sterilized, added after autoclaving).

2×YT ampicillin plates were composed per liter of 2×YT ampicillin mediumand 15 g of bacto agar.

LBS medium was composed of LB medium and 0.5 M sorbitol.

LBSM medium was composed of LBS medium and 0.38 M mannitol.

TBAB medium was composed of Difco Tryptose Blood Agar Base (BDDiagnostics, Franklin Lakes, N.J., USA).

TBAB chloramphenicol plates were composed of TBAB medium and 5 μg ofchloramphenicol per ml.

TBAB neomycin plates were composed of TBAB medium and 6 μg of neomycinper ml.

TBAB erythromycin/lincomycin plates were composed of TBAB medium and 1μg of erythromycin and 25 μg of lincomycin per ml.

TY medium was composed per liter of 20 g tryptone, 5 g yeast extract, 6mg FeCl₂.4H₂O, 1 mg MnCl₂.4H₂O, and 15 mg MgSO₄.7H₂O.

TY plates were composed of TY medium and 20 g of bacto agar per liter.

TY chloramphenicol plates were composed of TY plate medium containing 6μg chloramphenicol per ml.

LBPG plates were composed of LB plate medium containing 0.01 M K₃PO₄ and0.4% glucose.

Example 1 Determination of the Genome Sequence for Bacilluslicheniformis Strain SJ1904

The genome sequence for the entire chromosome of Bacillus licheniformisstrain SJ1904 was determined from contigs generated using 454 DNAsequencing technology (Margulies et al., 2005, Nature 437: 376-380),random paired reads using Sanger sequencing technology, and, to closegaps and resolve repeats, reads from PCR fragments of genomic DNA.Sequencing data was assembled using Phrap, and edited and viewed inConsed. Gene models were predicted from the genomic DNA sequence usingGlimmer (Delcher et al., 1999, Nucleic Acids Research 27: 4636-4641).Gene models were machine annotated by comparison to the nonredundantdatabase PIR-NREF (Wu et al., 2002, Nucleic Acids Research 30: 35-37)using a BLASTP with an E-value threshold of 1×10⁻⁵.

Example 2 Identification of Bacillus licheniformis M.Bli1904II DNAMethyltransferase Gene

The deduced amino acid sequences for the Bacillus licheniformis strainSJ1904 gene models were compared to the protein sequences from REBASE(Roberts, R. J., Macelis, M., Rebase. 2005) using BLASTP (Altschul etal., 1997, Nucleic Acids Research 25: 3389-3402). As the DNAmethyltransferases have a moderate level of sequence conservation, thisanalysis identified all putative DNA methyltransferases in this genome.A cytosine-specific DNA methyltransferase signature was identifiedwithin M.Bli1904II using Prints-S version 16 as implemented throughInterProScan release v3.3. In addition, the six highly conserved motifsfound in cytosine-specific DNA methyltransferases (Kumar et al., 1994,Nucleic Acids Research 22 1-10) were found to be conserved in theBacillus licheniformis M.Bli1904II DNA methyltransferase.

Example 3 Characterization of the Bacillus licheniformis M.Bli1904II DNAMethyltransferase Gene

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the Bacillus licheniformis M.Bli1904II DNAmethyltransferase gene are shown in FIGS. 1A and 1B. The coding sequenceis 1014 bp including the stop codon. The coding region is 36.1% G+C. Theencoded predicted protein is 337 amino acids with a molecular mass of38.5 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theBacillus licheniformis M.Bli1904II DNA methyltransferase shared 64%identity with a Bacillus weihenstephanensis C-5 cytosine-specific DNAmethyltransferase precursor (UniRef100_Q2AVE0) and shared 47% identitywith an Oceanobacillus iheyensis cytosine-specific DNA methyltransferase(UniRef100_Q8EL98). When the output of Needle labeled “longest identity”was used as the percent identity and was calculated as follows:

(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

the deduced amino acid sequence of the Bacillus licheniformisM.Bli1904II DNA methyltransferase shared 68.5% identity with theBacillus weihenstephanensis C-5 cytosine-specific DNA methyltransferaseprecursor (UniRef100_Q2AVE0) and 55.9% identity with the Oceanobacillusiheyensis cytosine-specific DNA methyltransferase (UniRef100_Q8EL98).

Example 4 Identification of the Bacillus licheniformis Bli1904II Type IIRestriction Endonuclease Gene

Type II restriction endonucleases generally share no sequence identitybetween one another and share only minor identity when they have similarDNA recognition sites. In addition, a Type II restriction endonucleaseis usually located next to its corresponding DNA methyltransferase in agiven restriction-modification system. Furthermore, all restrictionendonuclease genes characterized to date are larger than 450 bp (Kong,et al., 2000, Nucleic Acids Research 28: 3216-3223). Using thesecriteria, a hypothetical gene greater than 450 bp and located next to aBacillus licheniformis M.Bli1904II DNA methyltransferase gene wasidentified from the Bacillus licheniformis SJ1904 annotated gene modelsas a Type II restriction endonuclease gene, Bli1904II.

Example 5 Characterization of the Bacillus licheniformis Bli1904II TypeII Restriction Endonuclease Gene

The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the Bacillus licheniformis Bli1904II Type IIrestriction endonuclease gene are shown in FIGS. 2A and 2B. The codingsequence is 1146 bp including the stop codon. The coding region is 36.3%G+C. The encoded predicted protein is 381 amino acids with a molecularmass of 43.7 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Bacillus licheniformis Bli1904II Type 11 restrictionendonuclease shared 45% identity with a Bacillus weihenstephanensishypothetical protein (UniRef100_Q2AVE3) and 32.6% identity with aPsychromonas sp. CNPT3 hypothetical protein (UniRef100_Q1ZE16). When theoutput of Needle labeled “longest identity” was used as the percentidentity and was calculated as follows:

(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

the deduced amino acid sequence of the Bacillus licheniformisM.Bli1904II DNA methyltransferase shared 47% identity with the Bacillusweihenstephanensis hypothetical protein (UniRef100_Q2AVE3) and 47.2%identity with the Psychromonas sp. CNPT3 hypothetical protein(UniRef100_Q1ZE16).

Example 6 Cloning of the Bacillus licheniformis M.Bli1904II DNAMethyltransferase Gene

The Bacillus licheniformis M.Bli1904II DNA methyltransferase gene wascloned by PCR for expression in Bacillus subtilis.

Genomic DNA was isolated from Bacillus licheniformis SJ1904 according tothe procedure of Pitcher et al., 1989, Lett. Appl. Microbiol. 8:151-156. FIG. 3 shows the region of the Bacillus licheniformischromosome comprising the genes encoding Bli1904II restrictionendonuclease and M.Bli1904II DNA methyltransferase. An approximately1043 bp fragment of the Bacillus licheniformis SJ1904 chromosomeincluding the ribosome binding site and coding region of the M.Bli1904IIDNA methyltransferase gene, comprising nucleotides 2019-3049 of SEQ IDNO: 5 (FIG. 3), was amplified by PCR from Bacillus licheniformis SJ1904genomic DNA using primers 999611 and 999612 shown below. Primer 999611incorporates a Sac I restriction site, and primer 999612 incorporates anMlu I restriction site.

Primer 999611: (SEQ ID NO: 6) 5′-GAGCTCTGCAAGGAGGTATAATTTTG-3′Primer 999612: (SEQ ID NO: 7) 5′-ACGCGTTTATTCAGCTATTGCATATTC-3′

The PCR was performed using Pfx PLATINUM® DNA Polymerase (Invitrogen,Carlsbad, Calif., USA). The amplification reaction (50 μl) was composedof 1×Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 1 mMMgSO₄, 300 μM of each dNTP, 0.3 μM of each primer, 1.25 units ofPLATINUM® Pfx DNA Polymerase, and approximately 200 ng of template DNA.The reaction was performed using a ROBOCYCLER® 40 Temperature Cycler(Stratagene Corporation, La Jolla, Calif., USA) programmed for 1 cycleat 95° C. for 2 minutes; 30 cycles each at 95° C. for 1 minute, 55° C.for 1 minute, and 68° C. for 1 minute; and 1 cycle at 68° C. for 3minutes.

The resulting PCR product of approximately 1043 bp was cloned intovector pCR4Blunt using a ZERO BLUNT® ® PCR Cloning Kit for Sequencing(Invitrogen, Carlsbad, Calif., USA) and transformed into ONE SHOT® TOP10Chemically Competent E. coli cells according to the manufacturer'sinstructions. Plasmid DNA was isolated from one transformant using aPlasmid Midi Kit (QIAGEN Inc., Valencia, Calif., USA) and confirmed bydigestions with Eco RI, Nco I, and Sna BI followed by 0.8% agaroseelectrophoresis in TBE (50 mM Tris base-50 mM boric acid-1 mM disodiumEDTA) buffer, which yielded expected fragments of 3939 bp and 1061 bpfor Eco RI; 3217 bp and 1783 bp for Nco I; and 4165 bp and 835 bp forSna BI. The DNA sequence of the cloned PCR fragment was confirmed by DNAsequencing. This plasmid was designated pMDT138 (FIG. 4).

Plasmid pMDT138 was transformed into E. coli XL1-Blue cells (StratageneCorporation, La Jolla, Calif., USA) according to the manufacturer'sinstructions, selecting for ampicillin resistance on 2×YT ampicillinplates at 37° C. One transformant was designated MDT45 and was depositedon Sep. 7, 2006, under the terms of the Budapest Treaty with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,and given accession number NRRL B-41967.

Example 7 Construction of pMDT100

Plasmid pMDT100 is an E. coli replicon containing theP_(amyL4199)/P_(short consensus amyQ)/P_(cryIIIA)/cryIIAstab tripletandem promoter driving expression of the Bacillus clausii alkalineprotease gene (aprH). This aprH expression cassette and the cat gene ofpC194 (Horinouchi and Weisblum, 1982, J. Bacteriol. 150: 804-814) areflanked on both sides by fragments of the Bacillus subtilisalpha-amylase (amyE) gene, permitting insertion of the aprH expressioncassette and cat gene at the amyE locus of the Bacillus subtilischromosome by double homologous recombination via the two amyEfragments. Replacement of the aprH gene in pMDT100 with another geneallows chromosomal insertion and expression of that gene in Bacillussubtilis. The construction of pMDT100 is described below.

Plasmid pNBT51. Plasmid pNBT10 (pDG268MCS-Pr_(cryIIIA)/cryIIIAstab/SAV;U.S. Pat. No. 6,255,076) was isolated from E. coli host DH5α, using aQIAGEN® Plasmid Kit (QIAGEN Inc., Valencia, Calif., USA) according tothe manufacturer's instructions, and digested with Cla I and Sca I.Cleavage occurred at the Cla I site at approximately codon 326 of theaprH coding sequence and not at the Cla I site at approximately codon23, which was blocked by methylation due to E. coli Dam DNAmethyltransferase. The Cla I ends were blunted using Klenow fragment(New England Biolabs, Inc., Beverly, Mass., USA) and dNTPs according tothe manufacturer's instructions. The digested plasmid was analyzed by0.8% agarose electrophoresis in TBE buffer, and a vector fragment ofapproximately 6615 bp was purified using a QIAQUICK® Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA). Plasmid pOS4301 (Bacillus GeneticStock Center, Ohio State University, Columbus, Ohio, USA) was digestedwith Sal I and Sca I, and the Sal I ends were blunted using Klenowfragment and dNTPs, as described above. The digested plasmid wasanalyzed by 0.8% agarose electrophoresis in TBE buffer, and a fragmentof approximately 840 bp bearing the E. coli rrnB transcriptionterminator was purified using a QIAQUICK® Gel Extraction Kit. The same840 bp Sal I/Sca I fragment could be isolated from the vector pKK223-3(GE Healthcare, Piscataway, N.J., USA) (FIG. 5). The pNBT10 vectorfragment and terminator-bearing fragment were ligated together with T4DNA ligase (Roche Diagnostics Corporation, Indianapolis, Ind., USA)according to the manufacturer's instructions, and E. coli DH5α (GibcoBRL, Gaithersburg, Md., USA) was transformed with the ligation accordingto the manufacturer's instructions, selecting for ampicillin resistanceon 2×YT ampicillin plates at 37° C. The resulting plasmid was designatedpNBT51 (pDG268-P_(cryIIIA)/cryIIAstab/SAVΔ) (FIG. 6).

Plasmid pNBT52. Plasmid pNBT51 was digested with Sfi 1, and the endswere blunted by incubation for 20 minutes at 11° C. with T4 DNApolymerase (Roche Diagnostics Corporation, Indianapolis, Ind., USA) and25 μM of each dNTP, followed by heat-inactivation of the polymerase byincubation for 10 minutes at 75° C. The blunt-ended plasmid was thendigested with Dra III and analyzed by 0.8% agarose electrophoresis inTBE buffer, and a vector fragment of approximately 5920 bp was purifiedusing a QIAQUICK® Gel Extraction Kit. Plasmid pNBT20(pDG268MCS-P_(short consensus amyQ)/SAV; U.S. Pat. No. 6,255,076) wasdigested with Dra III and Ed 13611, and a fragment of approximately 1641bp bearing a short consensus amyQ promoter (P_(short consensus amyQ))was purified using a QIAQUICK® Gel Extraction Kit. The pNBT51 vectorfragment and P_(short consensus amyQ) fragment were ligated as describedabove, and E. coli DH5α was transformed with the ligation as describedabove, selecting for ampicillin resistance on 2×YT ampicillin plates at37° C. Plasmid DNA was isolated from several transformants using aQIAPREP® 8 Miniprep Kit (QIAGEN, Valencia, Calif., USA), digested withSph I, and analyzed by 0.8% agarose electrophoresis in TBE buffer. Oneplasmid with expected restriction fragments of approximately 4873 bp and2688 bp was designated pNBT52(pDG268-P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAVΔ) (FIG. 7).

Plasmid pNBT53. Plasmid pNBT6 (pHP13amp-SAV; U.S. Pat. No. 6,255,076)was digested with Sfi I and Sac I and analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a vector fragment of approximately6438 bp was purified using a QIAQUICK® Gel Extraction Kit. PlasmidpNBT52 was digested with Sfi I and Sac I and analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a fragment of approximately 727 bpbearing the P_(short consensus amyQ)/P_(cryIIIA)/cryIIAstab tandempromoter was purified using a QIAQUICK® Gel Extraction Kit. The pNBT6vector fragment and P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAStabfragment were ligated as described above, and E. coli DH5α cells weretransformed with the ligation as described above, selecting forampicillin resistance on 2×YT ampicillin plates at 37° C. Plasmid DNAwas isolated from several transformants using a QIAPREP® 8 Miniprep Kit,digested with Pvu II, and analyzed by 0.8% agarose electrophoresis usingTBE buffer. One plasmid with expected restriction fragments ofapproximately 4903 bp, 1320 bp, and 942 bp was designated pNBT53(pHP13amp-P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAV) (FIG.8).

Plasmid pNBT54. Plasmid pNBT1 (pDG268MCS; U.S. Pat. No. 6,255,076) wasdigested with Sfi I and Bam HI and analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a vector fragment of approximately6040 bp was purified using a QIAQUICK® Gel Extraction Kit. PlasmidpNBT53 was digested with Sfi I and Bam HI and analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a fragment of approximately 1953 bpbearing the P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAVcassette was purified using a QIAQUICK® Gel Extraction Kit. The pNBT1vector fragment and P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAVfragment were ligated as described above, and E. coli DH5α cells weretransformed with the ligation as described above, selecting forampicillin resistance on 2×YT ampicillin plates at 37° C. Plasmid DNAwas isolated from several transformants using a QIAPREP® 8 Miniprep Kitand analyzed by simultaneous digestion with Sfi I and Bam HI followed by0.8% agarose gel electrophoresis in TBE buffer. One plasmid withexpected restriction fragments of approximately 6040 bp and 1953 bp wasdesignated pNBT54(pDG268MCS-P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAV) (FIG.9).

Plasmid pNBT35. Plasmid pNBT2 (pDG268MCSΔ-Pr_(cryIIIA)/cryIIIAstab/SAV;U.S. Pat. No. 6,255,076) was digested with Sfi I and Bam HI and analyzedby 0.8% agarose gel electrophoresis in TBE buffer, and a vector fragmentof approximately 5394 bp was purified using a QIAQUICK® Gel ExtractionKit. Plasmid pNBT54 was digested with Sfi I and Bam HI, and analyzed by0.8% agarose gel electrophoresis in TBE buffer, and a fragment ofapproximately 1953 bp bearing theP_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAV cassette waspurified using a QIAQUICK® Gel Extraction Kit. The pNBT2 vector fragmentand P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab/SAV fragment wereligated as described above, and E. coli DH5α cells were transformed withthe ligation as described above, selecting for ampicillin resistance on2×YT ampicillin plates at 37° C. Plasmid DNA was isolated from severaltransformants using a QIAPREP® 8 Miniprep Kit, digested with Nco I, andanalyzed by 0.8% agarose gel electrophoresis in TBE buffer. One plasmidwith expected restriction fragments of approximately 5492 bp and 1855 bpwas designated pNBT35(pDG268MCSΔ-P_(short consensus amyQ)/P_(cryIIIA)/CryIIIAstab/SAV) (FIG.10).

Plasmid pNBT30. Plasmid pNBT30 was constructed to contain a PCR clone ofthe amyL4199 variant of the amyL gene promoter (U.S. Pat. No.6,100,063). Bacillus licheniformis SJ1904 genomic DNA was isolatedaccording to the procedure of Pitcher et al., 1989, supra. The amyL4199promoter (P amyL4199) amyL4199) gene was amplified by PCR from Bacilluslicheniformis SJ1904 genomic DNA using primers 950872 and 991151 shownbelow. Primer 950872 incorporates an Sfi I restriction site, and primer991151 incorporates a Sac I restriction site and the variant nucleotidesof P_(amyL4199).

Primer 950872: (SEQ ID NO: 8)5′-CCAGGCCTTAAGGGCCGCATGCGTCCTTCTTTGTGCT-3′ Primer 991151:(SEQ ID NO: 9) 5′-GAGCTCCTTTCAATGTGATACATATGA-3′

The PCR was performed using AMPLITAQ® Gold DNA Polymerase (AppliedBiosystems, Foster City, Calif., USA) according to manufacturer'srecommendations, except that the MgCl₂ concentration was 3 mM, ratherthan the standard 1.5 mM. The amplification reaction (50 μl) wascomposed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.0 mM MgCl₂, 200 μM ofeach dNTP, 0.5 μM of each primer, 0.25 units of AMPLITAQ® Gold DNAPolymerase, and approximately 200 ng of template DNA. The PCR wasperformed in a ROBOCYCLER® 40 Temperature Cycler programmed for 1 cycleat 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute, 55° C.for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 3minutes.

The resulting PCR product of approximately 625 bp was cloned into vectorpCR2.1 using a TOPO® TA Cloning Kit (Invitrogen, Carlsbad, Calif., USA)and transformed into ONE SHOT® TOP10 Chemically Competent E. coli cells(Invitrogen, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. Plasmid DNA was isolated from several transformants usinga QIAPREP® 8 Miniprep Kit and analyzed for the presence of the clonedPCR fragment by digestion with Eco RI followed by 0.8% agaroseelectrophoresis in TBE buffer. One plasmid with expected restrictionfragments of approximately 3913 bp and 640 bp was designated pNBT30(pCR2.1-amyL4199) (FIG. 11). The DNA sequence of the cloned PCR fragmentwas confirmed by DNA sequencing.

Plasmid pNBT31. Plasmid pNBT3(pDG268MCSΔneo-Pr_(cryIIIA)/cryIIIAstab/SAV; U.S. Pat. No. 6,255,076)was digested with Sfi I and Sac I and analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a vector fragment of approximately7931 bp was purified using a QIAQUICK® Gel Extraction Kit. PlasmidpNBT30 was digested with Sfi I and Sac I, analyzed by 0.8% agaroseelectrophoresis in TBE buffer, and a fragment of approximately 612 bpbearing P_(amyL4199) was purified using a QIAQUICK® Gel Extraction Kit.The pNBT3 vector fragment and P_(amyL4199) fragment were ligated asdescribed above, and E. coli XL1-Blue cells (Stratagene Corporation, LaJolla, Calif., USA) were transformed with the ligation according to themanufacturer's instructions, selecting for ampicillin resistance on 2×YTampicillin plates at 37° C. Plasmid DNA was isolated from severaltransformants using a QIAPREP® 8 Miniprep Kit, digested with Nco I, andanalyzed by 0.8% agarose electrophoresis in TBE buffer. One plasmid withexpected restriction fragments of approximately 6802 bp and 1741 bp wasdesignated pNBT31 (FIG. 12).

Plasmid pNBT36. Plasmid pNBT35 was digested with Sfi I, and the endswere blunted using T4 DNA polymerase and dNTPs, as described above. Theblunt ended plasmid was then digested with Dra III, and analyzed by 0.8%agarose electrophoresis in TBE buffer. A vector fragment ofapproximately 5808 bp was purified using a QIAQUICK® Gel Extraction Kit.Plasmid pNBT31 was digested with Dra III and Ecl 13611, analyzed by 0.8%agarose electrophoresis in TBE buffer, and a fragment of approximately2150 bp bearing P_(amyL4199) was purified using a QIAQUICK® GelExtraction Kit. The pNBT35 vector fragment and P_(amyL4199) fragmentwere ligated as described above, and E. coli SURE® cells (StratageneCorporation, La Jolla, Calif., USA) were transformed with the ligationaccording to the manufacturer's instructions, selecting for ampicillinresistance on 2×YT ampicillin plates at 37° C. Plasmid DNA was isolatedfrom several transformants using a QIAPREP® 8 Miniprep Kit, digestedwith Nco 1, and analyzed by 0.8% agarose electrophoresis in TBE buffer.One plasmid with expected restriction fragments of approximately 5492 bpand 2466 bp was designated pNBT36 (FIG. 13).

Plasmid pMDT100. Plasmid pNBT13(pDG268Δneo-P_(amyL)/P_(cryIIIA)/cryIIIAstab/SAV; U.S. Pat. No.6,255,076) was digested with Dra III and Sac I, and a vector fragment ofapproximately 6395 bp was purified using a QIAQUICK® Gel Extraction Kit.Plasmid pNBT36 was digested with Dra III and Sac I, analyzed by 0.8%agarose electrophoresis in TBE buffer, and a fragment of approximately2873 bp bearing the P_(amyL4199)/P_(amyQ(sc))/P_(cryIIIA) triple tandempromoter was purified using a QIAQUICK® Gel Extraction Kit. The pNBT13vector fragment and P_(amyL4199)/P_(amyQ(sc))/P_(cryIIIA) fragment wereligated as described above, and E. coli SURE® cells were transformedwith the ligation as described above, selecting for ampicillinresistance on 2×YT ampicillin plates at 37° C. Plasmid DNA was isolatedfrom several transformants using a QIAPREP® 8 Miniprep Kit, digestedwith Apa I, and analyzed by 0.8% agarose electrophoresis in TBE buffer.One plasmid with expected restriction fragments of approximately 4974 bpand 4294 bp was designated pMDT100 (FIG. 14).

Example 8 Expression of the Bacillus licheniformis M.Bli1904II DNAmethyltransferase gene in Bacillus subtilis

The Bacillus licheniformis M.Bli1904II DNA methyltransferase gene wasinserted into the chromosome of Bacillus subtilis in order to expressthe DNA methyltransferase in that host, thereby allowing methylation ofDNA in Bacillus subtilis.

Plasmid pMDT100 was digested with Sac I and Mlu I and analyzed by 0.8%agarose electrophoresis in TBE buffer, and a vector fragment ofapproximately 8100 bp was purified using a QIAQUICK® Gel Extraction Kit.Plasmid pMDT138 was digested with Sac I and Mlu I, and a fragment ofapproximately 1033 bp bearing the M.Bli1904II gene was purified using aQIAQUICK® Gel Extraction Kit. The pMDT100 vector fragment andM.Bli1904II gene fragment were ligated as described above. This ligationplaced the M.Bli1904II gene downstream of theP_(amyL4199)/P_(short consensus amyQ)/P_(cryIIIA)/cryIIIAstab promoterand upstream of the aprH transcription terminator. Bacillus subtilis168Δ4 was transformed with the ligation according to the procedure ofAnagnostopoulos and Spizizen, 1961, J. Bacteriol. 81: 741-746 andtransformants were selected for chloramphenicol resistance on TBABchloramphenicol plates at 37° C. Chloramphenicol-resistant transformantswere screened for neomycin sensitivity on TBAB neomycin plates at 37° C.to determine whether the DNA had inserted into the amyE gene of theBacillus subtilis chromosome by double crossover.

The presence of the M.Bli1904II DNA methyltransferase expressioncassette at the amyE locus was confirmed by PCR using primers 994112 and999592 shown below (which bind within the triple tandem promoter andM.Bli1904II DNA methyltransferase gene, respectively) and primers 999611and 960456 shown below (which bind within the M.Bli1904II DNAmethyltransferase gene and amyE gene, respectively). One suchtransformant, containing the cat gene and the M.Bli1904II DNAmethyltransferase expression cassette at the amyE locus, was designatedBacillus subtilis MDT101.

Primer 994112: (SEQ ID NO: 10) 5′-GCGGCCGCTCGCTTTCCAATCTGA-3′Primer 999592: (SEQ ID NO: 11) 5′-ATCGATCAGCTTGGATAAACCCTA-3′Primer 999611: (SEQ ID NO: 12) 5′-GAGCTCTGCAAGGAGGTATAATTTTG-3′Primer 960456: (SEQ ID NO: 13) 5′-CGTCGACGCCTTTGCGGTAGTGGTGCTT-3′

The PCRs were performed using Taq DNA Polymerase (New England Biolabs,Inc., Beverly, Mass., USA) according to the manufacturer's instructions.The amplification reactions (50 μl) were composed of 10 mM Tris-HCl (pH8.3), 50 mM KCl, 3.0 mM MgCl₂, 200 μM of each dNTP, 0.5 μM of eachprimer, 0.25 units of Taq DNA Polymerase, and approximately 200 ng ofgenomic DNA. The PCRs were performed in a ROBOCYCLER® 40 TemperatureCycler programmed for 1 cycle at 95° C. for 2 minutes; 30 cycles each at95° C. or 2 minutes, 55° C. or 2 minutes, and 72° C. for 2 minutes; and1 cycle at 72° C. for 3 minutes.

In order to confirm that the cloned M.Bli1904II DNA methyltransferasewas able to methylate DNA, plasmid DNA was isolated from Bacillusstrains expressing the methyltransferase and tested for DNA methylation.Bacillus subtilis strains 168Δ4 and MDT101 were transformed with plasmidpCJ791 (U.S. Published Application 20030175902) according to theprocedure of Anagnostopoulos and Spizizen, 1961, supra, selecting forerythromycin resistance on TBAB erythromycin/lincomycin plates at 34° C.Plasmid pCJ791 DNA was isolated from one transformant each of Bacillussubtilis 168Δ4 and Bacillus subtilis MDT101, using a QIAGEN® PlasmidMidi Kit.

Bacillus licheniformis SJ1904 was transformed with pCJ791 DNA from theBacillus subtilis MDT101 transformant by electroporation according tothe procedure of Xue et al., 1999, J. Microbiol. Methods 34(3): 183-191.Briefly, 1-5 ml of an overnight culture of Bacillus licheniformis grownin LBS medium was used to inoculate 50 ml of fresh LBS medium, and theculture was incubated at 37° C. and 250 rpm. The culture was grown tostationary phase, and cells were harvested by centrifugation at 6500×gwhen the culture experienced an increase in growth rate after a periodof slow growth (1-3 hours after the end of exponential growth). Cellswere washed twice with 50 ml of ice-cold MSG (0.5 M mannitol, 0.5 Msorbitol, 10% glycerol) and resuspended in approximately 750 μl of MSG.Cells were transformed as follows or stored at −20° C. Sixty μl ofelectrocompetent cells were mixed with plasmid DNA in an electroporationcuvette with a 1-mm electrode gap and subjected to an electrical pulseusing a GENE PULSER® (Bio-Rad Laboratories, Inc., Hercules, Calif., USA)set to 25 μF, 200Ω, and 1.0 kV. Electroporated cells were thentransferred to 950 μl of LBSM medium containing 0.2 μg of erythromycinper ml for induction of erythromycin resistance. The transformants wereincubated for 2.5-3 hours at 34° C. and 250 rpm and then selected forerythromycin resistance on TBAB erythromycin/lincomycin plates at 34° C.

Plasmid pCJ791 DNA was isolated from one Bacillus licheniformis SJ1904transformant, using a QIAGEN® Plasmid Midi Kit. Plasmid pCJ791 DNAisolated from Bacillus subtilis 168Δ4, Bacillus subtilis MDT101, andBacillus licheniformis SJ1904 was digested with Fnu 4HI and with Sac I.Plasmid pCJ791 has 12 Fnu 4HI recognition sites and three Sac Irecognition sites. Fnu 4HI, which cleaves DNA at the sequence GCNGC, wasable to digest pCJ791 plasmid DNA from Bacillus subtilis 168Δ4 but notpCJ791 plasmid DNA from Bacillus subtilis MDT101 or Bacilluslicheniformis SJ1904, indicating that plasmid DNA from the latter twosources was methylated at the GCNGC sites. Sac 1, which cleaves DNA atthe sequence GAGCTC and is thus unaffected by methylation of thesequence GCNGC, was able to cleave pCJ791 plasmid DNA from all threesources.

Example 9 Cloning of the Bacillus licheniformis Bli1904II RestrictionEndonuclease Gene

The Bacillus licheniformis Bli1904II restriction endonuclease codingregion was cloned by PCR. Bacillus licheniformis SJ1904 genomic DNA wasisolated according to the procedure of Pitcher et al., 1989, supra. FIG.3 shows the region of the Bacillus licheniformis chromosome comprisingthe genes encoding the Bli1904II restriction endonuclease andM.Bli1904II DNA methyltransferase. An approximately 1158 bp fragment ofthe Bacillus licheniformis SJ1904 chromosome including the coding regionof the Bli1904II gene, comprising nucleotides 443-1588 of SEQ ID NO: 5(FIG. 3), was amplified by PCR from Bacillus licheniformis SJ1904genomic DNA using primers 060625 and 060626 shown below. Primer 060625incorporates a Kpn I restriction site, and primer 060626 incorporates aBam HI restriction site.

Primer 060625: (SEQ ID NO: 14) 5′-GGTACCATGTTCTATACTAATCAACC-3′Primer 060626: (SEQ ID NO: 15) 5′-GGATCCTTATTTGTTTTCATTTTCAA-3′

The PCR was performed using Pfx DNA Polymerase (Invitrogen, Carlsbad,Calif., USA). The amplification reaction (50 μl) was composed of 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 1.5 mM MgSO₄,300 μM of each dNTP, 0.3 μM of each primer, 1.25 units of PLATINUM® PfxDNA Polymerase, and approximately 200 ng of template DNA. The reactionwas performed in a ROBOCYCLER® 40 Temperature Cycler programmed for 1cycle at 95° C. for 2 minutes; 30 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 68° C. for 1 minute; and 1 cycle at 68° C. for3 minutes.

The resulting PCR product of approximately 1158 bp was cloned intopCR4Blunt using a Zero Blunt® TOPO® PCR Cloning Kit for Sequencing(Invitrogen, Carlsbad, Calif., USA) and transformed into ONE SHOT® TOP10Chemically Competent E. coli cells according to the manufacturer'sinstructions. Plasmid DNA was isolated from one transformant using aQIAGEN® Plasmid Midi Kit and confirmed by digestion with Eco RI followedby 0.8% agarose electrophoresis in TBE buffer, which yielded expectedfragments of 3939 bp, 897 bp, and 279 bp. The DNA sequence of the clonedPCR fragment was confirmed by DNA sequencing. This plasmid wasdesignated pMDT156 (FIG. 15), and the E. coli TOP10 transformantcontaining the plasmid was designated MDT46. MDT46 was deposited on Sep.7, 2006, under the terms of the Budapest Treaty with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, 1815 University Street, Peoria, Ill., 61604, and given theaccession number NRRL B-41968.

Example 10 Construction of a Deleted Version of the Bacilluslicheniformis

Bli1904II restriction endonuclease gene

A deleted version of the Bacillus licheniformis Bli1904II restrictionendonuclease gene was constructed by PCR to permit deletion of thenative gene in Bacillus licheniformis.

Genomic DNA was isolated from Bacillus licheniformis SJ1904 according tothe procedure of Pitcher et al., 1989, supra, and two fragments from theBli1904II chromosomal locus were amplified by PCR.

An approximately 505 bp fragment of the Bacillus licheniformis SJ1904chromosome upstream of the Bli1904II gene, comprising nucleotides1616-2102 of SEQ ID NO: 5, was amplified by PCR from Bacilluslicheniformis SJ1904 genomic DNA using primers 999592 and 999593 shownbelow. Primer 999592 incorporates a Cla I restriction site.

Primer 999592: (SEQ ID NO: 16) 5′-ATCGATCAGCTTGGATAAACCCTA-3′Primer 999593: (SEQ ID NO: 17) 5′-TTCACAAGATCTATTTCTTCTTTCAGACCC-3′

The PCR was performed using PLATINUM® Pfx DNA Polymerase, as describedin Example 6.

An approximately 507 bp fragment of the Bacillus licheniformis SJ1904chromosome including the last 42 codons of the Bli1904II gene plusdownstream DNA, comprising nucleotides 1-487 of SEQ ID NO: 5, wasamplified from Bacillus licheniformis SJ1904 genomic DNA using primers999594 and 999595. Primer 999594 incorporates a Not I restriction site.

Primer 999594 (SEQ ID NO: 18) 5′-AGAAATAGATCTTGTGAAATGGGTTCTTAT-3′Primer 999595: (SEQ ID NO: 19) 5′-GCGGCCGCTCATGTTCCCATATTCTT-3′

The PCR was performed using PLATINUM® Pfx DNA Polymerase, as describedin Example 6, except that the MgSO₄ concentration was 1.5 mM.

Primer 999593 (used in the upstream amplification) and primer 999594(used in the downstream amplification) have complementary ends.Therefore, the two amplifications had complementary ends that wouldpermit fusion of the two PCR fragments, as described below. Primers wereremoved from the two amplifications using a QIAQUICK® PCR PurificationKit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer'sinstructions. The two amplifications were used as template DNA for athird PCR, using primers 999592 and 999595, which fused the twofragments via the complementary ends provided by the sequences ofprimers 999593 and 999594. The PCR was performed using PLATINUM® Pfx DNAPolymerase, as described in Example 6, except that the template DNAconsisted of 2 μl of each of the two amplifications.

The resulting approximately 994 bp PCR product was purified using aQIAQUICK® Gel Extraction Kit and cloned into vector pCR4Blunt using aZero Blunt®TOPO® PCR Cloning Kit for Sequencing, and transformed intoONE SHOT® TOP10 Chemically Competent E. coli cells as described inExample 6. Plasmid DNA from several transformants was purified using aBIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA) and tested for thepresence and orientation of the cloned PCR fragment by digestion withEco RI and with Not I followed by 0.8% agarose electrophoresis in TBEbuffer. One plasmid with Eco RI fragments of approximately 3939 bp and1012 bp and a Not I fragment of approximately 4931 bp was designatedpMDT134 (FIG. 16). The DNA sequence of the cloned PCR fragment wasconfirmed by DNA sequencing.

Example 11 Construction of Bacillus licheniformis Bli1904II RestrictionEndonuclease Gene Deletion Plasmid

A temperature-sensitive plasmid was constructed to allow deletion of theBli1904II restriction endonuclease gene in Bacillus licheniformis.

Plasmid pMDT131 was constructed to create a temperature-sensitiveplasmid conferring chloramphenicol resistance. Plasmid pMRT074 (U.S.Published Application 2003/0175902) was digested with Eco RI and thentreated with T4 DNA polymerase plus dNTPs to generate blunt ends, asdescribed in Example 7. The plasmid was then digested with Not 1,analyzed by 0.8% agarose electrophoresis in TBE buffer, and a vectorfragment of approximately 4355 bp was purified using a QIAQUICK® GelExtraction Kit. Plasmid pNBT1 was digested with Eco 47111 and Not 1,analyzed by 0.8% agarose electrophoresis in TBE buffer, and a fragmentof approximately 1222 bp bearing the cat gene and a multiple cloningsite was purified using a QIAQUICK® Gel Extraction Kit. The pMRT074vector fragment was ligated with the pNBT1 cat fragment using T4 DNAligase as described above, and Bacillus subtilis 168Δ4 was transformedwith the ligation according to the procedure of Anagnostopoulos andSpizizen, 1961, supra, selecting for chloramphenicol resistance on TBABchloramphenicol plates at 34° C. Plasmid DNA was isolated from onetransformant using a QIAGEN® Plasmid Midi Kit and confirmed by digestionwith Bam HI followed by 0.8% agarose electrophoresis in TBE buffer,which yielded expected fragments of approximately 3779 bp and 1802 bp.The resulting plasmid was designated pMDT131 (FIG. 17).

Plasmid pMDT139 was constructed to create a plasmid suitable fordeletion of the Bli1904II gene from the chromosome of Bacilluslicheniformis. E. coli SCS110 (Stratagene Corporation, La Jolla, Calif.,USA), which is deficient for DNA methyltransferase Dam, was transformedwith plasmid pMDT134 according to the manufacturer's instructions,selecting for ampicillin resistance on 2×YT ampicillin plates at 37° C.Plasmid DNA was isolated from one transformant using a QIAGEN® PlasmidMidi Kit, resulting in pMDT134 plasmid DNA that can be digested with ClaI (which in some contexts is inhibited by Dam methylation). PlasmidpMDT134 DNA was digested with Cla I and Not I and analyzed by 0.8%agarose electrophoresis in TBE buffer, and a fragment of approximately986 bp bearing the deleted Bli1904II gene was purified using a QIAQUICK®Gel Extraction Kit. Plasmid pMDT131 was digested with Cla I and Not I,analyzed by 0.8% agarose electrophoresis in TBE buffer, and a vectorfragment of approximately 5569 bp was purified using a QIAQUICK® GelExtraction Kit. The pMDT131 vector fragment was ligated with the deletedBli1904II gene fragment using T4 DNA ligase as described above, andBacillus subtilis 168Δ4 was transformed with the ligation according tothe procedure of Anagnostopoulos and Spizizen, 1961, supra, selectingfor chloramphenicol resistance on TBAB chloramphenicol plates at 34° C.Plasmid DNA was isolated from one transformant using a QIAGEN® PlasmidMidi Kit and confirmed by digestion with Pvu II, which yielded expectedfragments of approximately 4334 bp and 2220 bp. The resulting plasmidwas designated pMDT139 (FIG. 18).

Example 12 Deletion of the Bli1904II Gene in Bacillus licheniformisSJ1904

The Bacillus licheniformis Bli1904II restriction endonuclease gene wasdeleted from the chromosome of Bacillus licheniformis SJ1904 in order totest the effect on introduction of DNA into Bacillus licheniformis.

The Bli1904II gene was deleted from the chromosome of Bacilluslicheniformis SJ1904 as follows. Bacillus licheniformis SJ1904 wastransformed with plasmid pMDT139 bp electroporation according to theprocedure of Xue et al., 1999, supra, as described in Example 8. Thetransformants were incubated for 2.5-3 hours at 34° C. and 250 rpm andthen selected for erythromycin resistance on TBABerythromycin/lincomycin plates at 34° C.

One such transformant was grown on TBAB plates with erythromycinselection at 50° C. in order to select for integration of pMDT139 intothe chromosome at the Bli1904II locus. One such integrant was then grownin VY medium without selection at 34° C. in order to permit excision andloss of the integrated plasmid. The culture was plated on LB plates at37° C., and colonies were tested for sensitivity to erythromycin,indicating loss of the plasmid. Several erythromycin-sensitive cloneswere tested by PCR with primers 999592 and 999595 (shown above) using anEXTRACT-N-AMP™ Plant PCR Kit (Sigma-Aldrich, St. Louis, Mo., USA), asfollows. Bacillus licheniformis cells were lysed by suspending a colonyin 50 μl of Extraction Solution from the kit and incubating at 95° C.for 10 minutes. Each lysed suspension was then mixed with 50 μl ofDilution Solution from the kit, and 4 μl of each was used in a PCRaccording to manufacturer's instructions. One clone in which the PCRamplified a fragment of approximately 994 bp, indicating deletion of theBli1904II gene from the chromosome, was designated Bacilluslicheniformis MDT269.

Example 13 Transformation of Bacillus licheniformis Strains SJ1904 andMDT269

Transformation experiments were performed in order to determine theeffects of Bli1904II restriction endonuclease and methyltransferaseM.Bli1904II on introduction of DNA into Bacillus licheniformis.

Plasmid pCJ791 was isolated from transformants of Bacillus subtilis168Δ4, Bacillus subtilis MDT101, and Bacillus licheniformis SJ1904 asdescribed in Example 8. Electrocompetent cells of Bacillus licheniformisstrains SJ1904 and MDT269 were prepared as described in Example 8. BothBacillus licheniformis strains were transformed with pCJ791 plasmid DNAisolated from the three Bacillus sources. For each transformation, 60 μlof electrocompetent cells were transformed with 200 ng of plasmid DNA inan electroporation cuvette with a 1-mm electrode gap, using a GENEPULSER® set to 25 μF, 200Ω, and 1.0 kV. Transformations were performedin triplicate.

The results of the transformations are shown in Table 1. A lowtransformation frequency was obtained when Bacillus licheniformis SJ1904(with a wild-type Bli1904II restriction endonuclease gene) wastransformed with plasmid DNA from Bacillus subtilis 168Δ4 (notexpressing methyltransferase M.Bli1904II). However, high transformationefficiencies were obtained when Bacillus licheniformis SJ1904 wastransformed with plasmid DNA that was methylated by methyltransferaseM.Bli1904II (from Bacillus subtilis MDT101 or Bacillus licheniformisSJ1904). Furthermore, high transformation efficiencies were obtainedwhen Bacillus licheniformis MDT269 (with the Bli1904II restrictionendonuclease gene deleted) was transformed with plasmid DNA from any ofthe three sources (whether methylated by methyltransferase M.Bli1904IIor not). The results showed that transformation of Bacilluslicheniformis by electroporation with plasmid DNA was significantlyimproved either by isolating the plasmid DNA from a host strainexpressing M.Bli1904II DNA methyltransferase, which modifies the plasmidDNA, or by deleting the Bli1904II restriction endonuclease gene in therecipient Bacillus licheniformis strain.

TABLE 1 Transformation of Bacillus licheniformis SJ1904 and MDT269 withpCJ791 plasmid DNA isolated from Bacillus subtilis 168Δ4, Bacillussubtilis MDT101, and Bacillus licheniformis SJ1904. Bacilluslicheniformis Transformants strain Source of pCJ791 DNA per μg DNA¹ B.licheniformis Bacillus subtilis 168Δ4 17 ± 15 SJ1904 (nomethyltransferase (wild-type M.Bli1904II gene) Bli1904II gene) Bacillussubtilis MDT101 1.6 × 10⁴ ± 8.3 × 10³ (cloned methyltransferaseM.Bli1904II gene) Bacillus licheniformis SJ1904 1.2 × 10⁴ ± 5.7 × 10³(native methyltransferase M.Bli1904II gene) B. licheniformis Bacillussubtilis 168Δ4 6.4 × 10³ ± 9.6 × 10² MDT269 (no methyltransferase(deleted M.Bli1904II gene) Bli1904II gene) Bacillus subtilis MDT101 7.2× 10³ ± 2.1 × 10³ (cloned methyltransferase M.Bli1904II gene) Bacilluslicheniformis SJ1904 4.1 × 10³ ± 5.8 × 10² (native methyltransferaseM.Bli1904II gene) ¹Transformation frequency is the mean of threereplicates ± standard deviation.

Example 14 Transformation of Bacillus licheniformis with Plasmid DNAMethylated In Vitro

A cell-free extract (CFE) of Bacillus licheniformis SJ1904 was preparedaccording to the method of Alegre et al., 2004, FEMS MicrobiologyLetters 241: 73-77. Plasmid pCJ791 isolated from a transformant ofBacillus subtilis 168Δ4 was methylated by treatment with the CFE andS-adenosylmethionine (SAM), as described by Alegre et al., 2004, supra.In addition, a control was performed in which the CFE was replaced withthe buffer used to prepare the CFE. After treatment, the DNA wasextracted with phenol and chloroform, precipitated with isopropanol, andresuspended in 20 μl water. Bacillus licheniformis SJ1904 was thentransformed by electroporation as described in Example 8, using 200 ngof pCJ791 plasmid DNA, either treated as described above or untreated.Transformations were performed in triplicate.

The results of the transformations are shown in Table 2. Transformationwith plasmid DNA that had been treated with CFE plus SAM resulted inmore than 100-fold higher transformation frequency than transformationwith either untreated plasmid DNA or plasmid DNA treated with SAM alone.These results confirmed that methylation of plasmid DNA in vitroimproved transformation of Bacillus licheniformis.

TABLE 2 Transformation of Bacillus licheniformis SJ1904 with pCJ791plasmid DNA methylated in vitro. Plasmid DNA was isolated from Bacillussubtilis 168Δ4. Treatment of plasmid DNA Transformants per μg DNA¹ B.licheniformis SJ1904 CFE + SAM 6.9 × 10³ ± 1.2 × 10³ CFE buffer + SAM 30± 20 untreated 50 ± 20 ¹Transformation frequency is the mean of threereplicates ± standard deviation.

Example 15 Effect of Bacillus licheniformis Restriction-ModificationSystem on Conjugal Plasmid Transfer in Bacillus subtilis

The effect of the Bacillus licheniformis restriction-modification systemon conjugal plasmid transfer was tested using plasmid pCJ791, which maybe transferred by conjugation from a suitable donor cell to a recipientcell, due to the presence of the oriT origin of transfer present on theplasmid. One such suitable conjugation donor strain is Bacillus subtilisPP289-5 (WO 96/029418), which has a deletion in the arl (dal) geneencoding D-alanine racemase and contains plasmids pBC16 (conferringtetracycline resistance) and pLS20, which confer the ability to mobilizeoriT-containing plasmids.

Bacillus subtilis PP289-5 was transformed with genomic DNA isolated fromBacillus subtilis MDT101 according to the procedure of Anagnostopoulosand Spizizen, 1961, supra, selecting for chloramphenicol resistance onTY chloramphenicol plates at 37° C. One such transformant, containingthe cat gene and the M.Bli1904II DNA methyltransferase expressioncassette at the amyE locus, was designated Bacillus subtilis AEB711.

Bacillus subtilis strains PP289-5 and AEB711 were transformed withplasmid pCJ791 according to the procedure of Anagnostopoulos andSpizizen, 1961, supra, selecting for erythromycin resistance on TBABerythromycin/lincomycin plates at 34° C. One PP289-5 transformant wasdesignated Bacillus subtilis MDT104, and one AEB711 transformant wasdesignated Bacillus subtilis MDT105.

Bacillus subtilis donor strains MDT104 and MDT105 were used to transferplasmid pCJ791 to Bacillus licheniformis recipient strains SJ1904 andMDT269 bp conjugation. Bacillus licheniformis SJ1904 and MDT269 weregrown overnight at 30° C. on LBPG plates. Bacillus subtilis MDT104 andMDT105 were grown overnight at 30° C. on LBPG plates containing 100 μgof D-alanine, 10 μg of tetracycline, and 5 μg of erythromycin per ml.Cells of each strain were then suspended in TY medium, and the opticaldensity (OD) was measured at 450 nm. Based on OD, equal amounts of onedonor strain and one recipient strain were mixed, using the equivalentof 100 μl of a cell suspension having an (O)₄₅₀ of 50. Mixtures werespread on LB plates containing 100 pg of D-alanine per ml and incubatedat 30° C. Under these conditions, both the donor and recipient strainwere able to grow, and plasmid pCJ791 could be transferred byconjugation from donor to recipient. After incubation for 6 hours, cellswere suspended in 1 ml of TY medium, aliquots were spread on TY platescontaining 2 μg of erythromycin per ml, and the plates were incubatedovernight at 30° C. Under these conditions, only Bacillus licheniformistransconjugants (recipient cells that had received plasmid pCJ791 bpconjugal transfer) were able to grow. The absence of D-alanine preventedgrowth of the arl-negative donor, and the presence of erythromycinprevented growth of the original recipient.

The number of transconjugants resulting from each conjugation wascompared for the four different combinations of donor and recipientstrains. The results of the conjugations are shown in Table 3. A lowconjugation frequency was obtained when Bacillus subtilis MDT104(containing no M.Bli1904II DNA methyltransferase) was used as donor withBacillus licheniformis SJ1904 (with a wild-type Bli1904II restrictionendonuclease gene) as recipient. However, high conjugation frequencieswere obtained when Bacillus subtilis MDT105 (expressing M.Bli1904II DNAmethyltransferase) was used as donor (regardless of recipient) or whenBacillus licheniformis MDT269 (with the Bli1904II restrictionendonuclease gene deleted) was used as recipient (regardless of donor).The results showed that conjugation of Bacillus licheniformis wassignificantly improved either by using a donor strain expressingM.Bli1904II DNA methyltransferase, which modifies the plasmid DNA, or bydeleting the Bli1904II restriction endonuclease gene in the recipientBacillus licheniformis strain.

TABLE 3 Conjugal transfer of plasmid pCJ791 to Bacillus licheniformisstrains SJ1904 and MDT269 from Bacillus subtilis donor strains MDT104and MDT105. Number of transconjugants is listed for each of threeindependent experiments, A, B, and C. Bacillus licheniformis Bacillussubtilis Transconjugants recipient strain donor strain per experiment B.licheniformis SJ1904 Bacillus subtilis MDT104 A: 3300 (wild-typeBli1904II (no M.Bli1904II DNA B: 1730 restriction methyltransferasegene) C: 380 endonuclease gene) Bacillus subtilis MDT105 A: 500,000(cloned M.Bli1904II DNA B: 970,000 methyltransferase gene) C: 153,000 B.licheniformis Bacillus subtilis MDT104 A: 516,000 MDT269 (deleted (noM.Bli1904II DNA B: 1,170,000 Bli1904II restriction methyltransferasegene) C: 152,000 endonuclease gene) Bacillus subtilis MDT105 A: 957,000(cloned M.Bli1904II DNA B: 1,700,000 methyltransferase gene) C:1,350,000

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli MDT45 (pMDT138) NRRLB-41967 Sep. 7, 2006 E. coli MDT46 (pMDT156) NRRL B-41968 Sep. 7, 2006

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polynucleotide encoding a DNA methyltransferase selectedfrom: (a) a polynucleotide encoding a polypeptide comprising an aminoacid sequence having at least 95% sequence identity with amino acids 1to 337 of SEQ ID NO: 2; (b) a polynucleotide comprising a nucleotidesequence having at least 95% sequence identity with nucleotides 1 to1011 of SEQ ID NO: 1; and (c) a polynucleotide that hybridizes underhigh stringency conditions with nucleotides 1 to 1011 of SEQ ID NO: 1 orits full-length complementary strand.
 2. The isolated polynucleotide ofclaim 1, which encodes a DNA methyltransferase comprising an amino acidsequence having at least 95% sequence identity with amino acids 1 to 337of SEQ ID NO:
 2. 3. The isolated polynucleotide of claim 1, whichencodes a DNA methyltransferase comprising an amino acid sequence havingat least 97% sequence identity with amino acids 1 to 337 of SEQ ID NO:2.
 4. The isolated polynucleotide of claim 1, which encodes a DNAmethyltransferase comprising an amino acid sequence having at least 99%sequence identity with amino acids 1 to 337 of SEQ ID NO:
 2. 5. Theisolated polynucleotide of claim 1, which encodes a DNAmethyltransferase comprising or consisting of amino acids 1 to 337 ofSEQ ID NO:
 2. 6. The isolated polynucleotide of claim 1, comprising anucleotide sequence having at least 95% sequence identity withnucleotides 1 to 1011 of SEQ ID NO:
 1. 7. The isolated polynucleotide ofclaim 1, comprising a nucleotide sequence having at least 97% sequenceidentity with nucleotides 1 to 1011 of SEQ ID NO:
 1. 8. The isolatedpolynucleotide of claim 1, comprising a nucleotide sequence having atleast 99% sequence identity with nucleotides 1 to 1011 of SEQ ID NO: 1.9. The isolated polynucleotide of claim 1, comprising nucleotides 1 to1011 of SEQ ID NO:
 1. 10. The isolated polynucleotide of claim 1,comprising the DNA methyltransferase coding sequence contained inplasmid pMDT138 which is contained in Escherichia coli NRRL B-41967. 11.The isolated polynucleotide of claim 1, wherein the polynucleotidehybridizes under high stringency conditions with nucleotides 1 to 1011of SEQ ID NO: 1 or its full-length complementary strand.
 12. Theisolated polynucleotide of claim 1, wherein the polynucleotidehybridizes under very high stringency conditions with nucleotides 1 to1011 of SEQ ID NO: 1 or its full-length complementary strand.
 13. Anucleic acid construct or recombinant expression vector comprising thepolynucleotide of claim 1 operably linked to one or more controlsequences that direct the production of the DNA methyltransferase in anexpression host.
 14. The nucleic acid construct or recombinantexpression vector of claim 13, wherein the polynucleotide encodes a DNAmethyltransferase comprising an amino acid sequence having at least 95%sequence identity with amino acids 1 to 337 of SEQ ID NO:
 2. 15. Thenucleic acid construct or recombinant expression vector of claim 13,wherein the polynucleotide encodes a DNA methyltransferase comprising orconsisting of amino acids 1 to 337 of SEQ ID NO: 2
 16. A recombinanthost cell comprising the polynucleotide of claim
 1. 17. The recombinanthost cell of claim 16, wherein the polynucleotide encodes a DNAmethyltransferase comprising an amino acid sequence having at least 95%sequence identity with amino acids 1 to 337 of SEQ ID NO:
 2. 18. Therecombinant host cell of claim 16, wherein the polynucleotide encodes aDNA methyltransferase comprising or consisting of amino acids 1 to 337of SEQ ID NO:
 2. 19. A method of producing a DNA methyltransferase,comprising: (a) cultivating the recombinant host cell of claim 16 underconditions conducive for production of the DNA methyltransferase; and(b) recovering the DNA methyltransferase.
 20. A method of producingbacterial transformants, comprising: (a) introducing a DNA into a firstbacterial host cell comprising the polynucleotide of claim 1 encoding aDNA methyltransferase to methylate the DNA; (b) transferring themethylated DNA from the first bacterial host cell into a secondbacterial host cell, wherein the second bacterial host cell comprises arestriction endonuclease able to degrade the DNA but unable to degradethe methylated DNA; and (c) isolating transformants of the secondbacterial host cell comprising the methylated DNA.
 21. The method ofclaim 20, wherein the polynucleotide encodes a DNA methyltransferasecomprising an amino acid sequence having at least 95% sequence identitywith amino acids 1 to 337 of SEQ ID NO:
 2. 22. The method of claim 20,wherein the polynucleotide encodes a DNA methyltransferase comprising orconsisting of amino acids 1 to 337 of SEQ ID NO: 2.